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A BIOCHEMICAL STUDY OF NITROGEN 
IN CERTAIN LEGUMES 



BY 



ALBERT LEMUEL WHITING 

B. S. Massachusetts Agricultural College, 1908 

M. S. Rhode Island State College, 1910 



THESIS 
Submitted in Partial Fulfillment of the Requirements for the 

Degree of 
DOCTOR OF PHILOSOPHY 
IN AGRONOMY 

IN 

THE GRADUATE SCHOOL 

OF THE 

UNIVERSITY OF ILLINOIS 
1912 



A BIOCHEMICAL STUDY OF NITROGEN 
IN CERTAIN LEGUMES 



BY 



ALBERT LEMUEL WHITING 

B. S. Massachusetts Agricultural College, 1908 

M. S. Rhode Island State College, 1910 



THESIS 
Submitted in Partial Fulfillment of the Requirements for the 

Degree of 
DOCTOR OF PHILOSOPHY 
IN AGRONOMY 

IN 

THE GRADUATE SCHOOL 

OF THE 

UNIVERSITY OF ILLINOIS 
1912 






6"^ 



UNIVERSITY OF ILLINOIS 

Agricultural Experiment Station 



BULLETIN No. 179 



A BIOCHEMICAL STUDY OF NITROGEN IN 
CERTAIN LEGUMES 



By albert L. WHITING 




URBANA, ILLINOIS, MARCH, 1915 



Contents of Bulletin No. 179 

PAGii 

INTRODUCTION 471 

HISTORICAL 472 

BIOLOGICAL 475 

Infection 476 

Inoculation as It Occurs Under Field Conditions 480 

Growth of the Nodule 481 

BACTERIOLOGICAL 482 

Bacillus radicicola 483 

Growth and Endurance of B. radicicola 484 

Identity of B. radicicola 484 

Enzyme Production by B. radicicola 485 

Slime Production by B. radicicola 486 

Isolation of B. radicicola 486 

Dissemination of B. radicicola 486 

Fixation of Nitrogen Without the Legume Plant 487 

Bacteroids 487 

THEORIES OF ASSIMILATION, FIXATION, AND IMMUNITY 488 

Theories of Assimilation by the Plant 488 

Theories Regarding the Chemical Phenomena of Fixation 489 

Theories of Immunity 492 

PRACTICAL CONSIDERATIONS WITH REGARD TO LEGUME 

FIXATION 493 

Mutual Symbiosis 493 

Amount of Nitrogen Fixed per Acre per Year 495 

Value of Legumes as Nitrogen Retainers 496 

Cross Inoculation 497 

Associative Growth of Legumes and Non-Legumes 498 

CHEMICAL 499 

EXPERIMENTAL 503 

Plan of Investigations 503 

Part I: Studies to Determine Thru Which Organs Legumes Obtain 

Atmospheric Nitrogen 503 

General Plan of Experiments 505 

Experiment I 507 

Experiment II 507 

Experiment III 509 

Experiment IV 514 

Experiment by Kossowitsch 519 

General Consideration of Gas Experiments 521 

Practical Application of Results 522 



PAGE 

Paet II: Eelative Percentages of Nitrogenous Compounds in the 
Various Parts of the Soybean and Cowpea at Definite Periods 

OF Growth 522 

Methods Employed in the Growth and Preparation of Samples. .523 

Analytical Methods 525 

Discussion of Some of the Methods Used 527 

Qualitative Tests 527 

Series 100 (Soybeans) 528 

Series 500 (Soybeans) 532 

Series 700 (Soybeans) 533 

Series 600 (Cowpeas) 536 

Dis^cussion of Tables 537 

CONCLUSIONS 541 



Illustrations 

Plate page 
I.— Nodules of Robina Tyjie on Roots of Soybeans 477 

II. — Nodules of Lupinus Type on Roots of Lupine Seedlings 478 

III.— Nodules of Robina Type on (A) Red Clover; (B) Vetch; (C) 

Sweet Clover 479 

IV. — Cowpea Seedlings in Preparation for Gas Experiments 504 

V. — Experiment II: Cowpeas at Harvest (37 Days) 506 

VI. — Experiment II: Plants Grown in Air and in COj-j-0 508 

VII. — Experiment III: At Beginning and 10 Days Later 510 

VIII. — Experiment III: 52 Days from Beginning and at Harvest (83 

Days) 511 

IX. — Experiment III: Roots from Plants Grown in CO. -|- O and in Air. .513 

X. — Experiment IV: At Beginning and 16 Days Later 515 

XL — Experiment IV: 41 and 59 Days from Beginning 516 

XII.— Experiment IV : At Harvest (95 Days) 517 

XIII. — Experiment IV : Roots from Plants Grown in CO2 -j- O and in 

N + COo + 518 

XIV. — Typical Jar of Five Cowpeas Being Grown for Samples 523 

XV. — Graph Showing Soluble and Insoluble Nitrogen in Series 100 531 

XVI. — Graph Showing Soluble and Insoluble Nitrogen in Series 700 and 500.535 

XA-'II. — Graph Showing Soluble and Insoluble Nitrogen in Series 600 53S 

Figure 

1. — Root hair of common pea, showing infecting strand 476 

2. — Root cell, showing infecting strand passing thru it and the formation 
of lamellae 476 

3. — Young nodule magnified, showing affected root hair and same root 

hair more highly magnified 480 

4. — Young nodule, showing the beginning of the differentiation of its tissues. .482 

5. — B. radicieola, showing shajie and flagella 483 

6. — Bacteroids, showing shape, and occurrence of vacuoles 488 



A BIOCHEMICAL STUDY OF NITROGEN 
IN CERTAIN LEGUMES^ 

By ALBEET L. WHITING, Associate in Soil Biology 

INTRODUCTION 

The investigations considered in this publication bear on the 
biochemical nature of the element nitrogen, especially as concerns its 
fixation and assimilation thru the symbiotic relationship of Bacillus 
radicicola and certain members of the botanical family known as 
Leguminosae. 

The sources of the element nitrogen available for agricultural 
purposes are numerous. Of these the atmosphere is by far the most 
important and most extensive. Above each acre of the earth's surface 
there are about 69 million pounds of atmospheric nitrogen, and 
science has shown that by thoroly scientific systems of management 
this nitrogen may be appropriated for soil improvement at a minimum 
expense. By growing legumes, atmospheric nitrogen may be obtained 
at a low cost, often at no net cost, for most agricultural leguminous 
crops are worth growing for feed or seed alone. In commercial fer- 
tilizing materials, nitrogen costs from fifteen to twenty cents per 
pound, an amount from two to five times greater than that expended 
for any of the other essential elements of plant food. It is of passing 
interest to note how greatly disproportionate the cost values of these 
elements are to the relative supplies, when the nitrogen in the air is 
considered. 

The United States spends annually, abroad, over 32 million dol- 
lars in the purchase of combined nitrogen for use in various opera- 
tions, agricultural and otherwise.^ Of this amount I6I/2 million dol- 
lars are expended for the purchase of sodium nitrate, which is the 

^Submitted to the Faculty of the Graduate School of the University of Illi- 
nois in partial fulfilment of the requirements for the degree of doctor of phi- 
losophy, June, 1912. Revised to date of issuance. 

^Norton : Special Agent Series, Dept. of Commerce and Labor, Bur. of Manf r., 
No. 52, 9-11. 



471 



472 Bulletin No. 179 [March, 

most important commercial form of inorganic nitrogen. The present 
world supply of this salt is estimated at 454,576,200.000 pounds.^ 

How insufficient this supply is, when measured by crop require- 
ments, may be realized from the fact that the following nine important 
crops of the United States, — corn, wheat, oats, barley, rye, potatoes, 
hay, cotton, and tobacco, in the year 1910, required for their growth 
11,500,000,000 pounds of nitrogen.^ If sodium nitrate were used for 
growing these crops at the rate stated above, the supply would be ex- 
hausted in about six years. On the other hand, the nitrogen above 
only one square mile, weighing 20 million tons, would be sufficient 
to supply what the entire world, at its present rate of consumption, 
would require for the next fifty years.^ The nitrogen above four 
acres would furnish more than the actual yearly consumption of 
commercial nitrogen in the entire United States. 

The wonderful possibilities presented by such an extensive source 
of plant food, and the fact that over 100 million dollars are invested 
in commercial fertilizers each year in the United States, a large part 
of which is wasted or uselessly applied, together with the great natural 
losses of nitrogen that occur, tend to emphasize greatly the need of a 
proper utilization of this unlimited reserve supply. Further, it is well 
recognized that the maintenance of the nitrogen supply is the greatest 
of our soil problems. Nitrogen cannot be purchased on the market at 
a price that will permit its extensive application in growing the im- 
portant crops of the United States. There is only one logical and in- 
exhaustible source of nitrogen for the world to utilize in the produc- 
tion of crops. That source is the atmosphere, from which nitrogen 
is most economically and easily secured as a result of the symbiotic 
relationship between B. radicicola and leguminous plants. 

HISTORICAL 

For several centuries certain plants of the Leguminosae have been 
used as soil improvers. A few of the more important references to 
their uses are considered here. 

In Roman literature, among the works of Columella,'* mention is 
made of the Roman farmers regarding beans as possessing the prop- 
erty of enriching the soil, and attention is also called to the practice 
of plowing under lupines. Alfalfa and vetches were observed to pro- 
duce similar results to those of lupines and beans. Like notations 



^Eeview of Eeviews, April, 1910. 

^Yields taken from U. S. Yearbook, 1910. For calculations see Hopkins' 
"Soil Fertility and Permanent Agriculture" (1910), 154; also 603-604. 

'Norton: Special Agent Series, Dept. of Commerce and Labor, Bur. of Manfr., 
No. 52, 9-11. 

^Marshall: Microbiology (1911), 273. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 473 

may be found among the writings of Thaer and Walz.^ Gasparin^ 
constantly calls attention to the power of leguminous plants to add 
nitrogen to the soil. Jethro Tull-^ wrote concerning the efficiency of 
legumes in restoring depleted soils, mentioning especially sanfoin and 
alfalfa. 

It may be noted here that Hellriegel, who later was most promi- 
nent in the discovery of the relation existing between legumes and 
bacteria, wrote in 1863 as follows: "Clover plants may develop nor- 
mally and completely in mere sand to which the necessary mineral 
constituents of plant food have been added in assimilable forms, even 
when this soil contains no trace of any compound of nitrogen or of 
organic matter." 

Schultz-Lupitz^ in 1881 reported results that were of both chemi- 
cal and practical significance. After growing lupines for fifteen con- 
secutive times on a sandy soil, without the application of nitrogenous 
materials, he observed that the yields did not diminish ; and when he 
grew cereals on the same land after the lupines, he found that the 
yields of the grains were two and three times the yields where no 
lupines had been grown. Analyses of the soils at the end of this time 
showed that where the lupines had been grown, the nitrogen content 
of the surface six inches had increased by .06 percent. Frank^ veri- 
fied these results with twenty years of lupine culture on the same 
fields. 

About this time a great deal of interest centered on pot-culture 
experiments with legumes. Many physiologists and chemists worked 
on the problem of nitrogen collection by legumes. Prominent among 
these were the scientists Boussingault,^ and Lawes, Gilbert, and Pugh,'^ 
who, owing to their great accuracy, sacrificed the possibility of becom- 
ing the discoverers of this important relationship. In their great care, 
they destroyed the vital agency {B. radicicola) necessary for the ac- 
complishment of this symbiotic fixation. 

Later, in 1886, Hellriegel and his co-worker Wilfarth^ made the 
classical discovery that legumes obtain atmospheric nitrogen thru the 
association of microorganisms living in the nodules. In a preliminary 
report read before a section of scientists assembled on September 20, 
1886, at Berlin, Hellriegel announced his findings ; and in a more com- 



^Storer: Agriculture (1906), 2, 97. 
^Ibid. 

^Lipman: Bacteria in Relation to Country Life (1908), 206. 
'Schultz-Lupitz: Landw. Jahrb. (1881), 10, 777. 
"Frank: Landw. Jahrb. (1888), 17, 501. 

"Boussingault: Ann. Sci. Agron. (1909), 26, Ser. 3, 4, 102-130. 
'Lawes, Gilbert, and Pugh: Eothamsted Experiments (1905), 6-7. 
'Hellriegel and Wilfarth: Tagblatt d. Naturforscher Versamml. z. Berlin 
(1886), 290. 



474 Bulletin No. 179 [March, 

plete account rendered two years later, he made known to the world 
his researches. These are summarized as follows i^ 

1. The legumes differ fundamentally from the grains in 
their nutrition with respect to nitrogen. 

2. The grains (Gramineae) can satisfy their nitrogen 
need only by means of assimilable combinations existing in 
the soil, and their development is always in direct proportion 
to the provision of nitrogen which the soil places at their 
disposal. 

3. Outside the nitrogen of the soil, the legumes have at 
their service a second source from which they can draw in 
most abundant manner all the nitrogen which their nutrition 
demands to complete that lack when the first source is in- 
sufficient. 

4. That second source is the free nitrogen — the elemen- 
tary nitrogen of the atmosphere which is furnished to them. 

5. The legumes do not possess by themselves the faculty 
of assimilating the free nitrogen from the air; it is ab- 
solutely necessary that the vital action of microorganisms of 
the soil come to their aid in order to attain this result. 

6. In order that the nitrogen of the air can be made to 
serve the nutrition of the legumes, the sole presence of lower 
organisms in the soil is not sufficient; it is still necessary 
that certain among them enter into a symbiotic relationship 
with the plants. 

7. The nodules^ of the roots must not be considered as 
simple reservoirs of albuminoid substances; their relation 
to the assimilation of free nitrogen is that of cause to effect. 

Schloesing and Laurent^ after growing legumes in a confined at- 
mosphere, gave out the following direct evidence of the fixation of at- 
mospheric nitrogen. 

Atmospheric nitrogen introduced 

into culture vessel 2681.2 ccm. 

Atmospheric nitrogen withdrawn 2653.1 ccm. 

Amount of nitrogen assimilated 28.1 ccm. 

(=36.5 mg.) 

Nitrogen in the soil and crop 73.2 mg. 

Nitrogen in the soil and seed 32.6 mg. 

Nitrogen assimilated 40.6 mg. 

*Hellriegel and Wilfarth: Beil. Zert. d. Verins. fiir die Eubenzucker In- 
dustrie, Berlin, Nov., 1888; or Lafar Handbuch der technischen Mykologie (1904- 
06), 3, 31. 

"Nodules substituted for tubercles. 

'Schloesing and Laurent: Compt. Rend. Acad. Sci. (1890), 111, 750: (1892) 
115, 659, 732. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 475 

In addition to the scientists mentioned above, Atwater and Woods, 
Berthelot, Miintz, Ville, Maze, Deherain, Frank, Hartig, Nobbe, Hilt- 
ner, Warrington, Hopkins, and many others have done much careful 
work in solving the problem and applying the truths discovered. 

BIOLOGICAL 

Nodules,^ which are the visible manifestations of infection, were 
observed upon the roots of legumes by Malphighi^ as early as 1687. 
The investigators of those times believed that the nodules were the re- 
sult of pathological processes, — that they were lumps, knobs, warts, 
and even galls. In 1853 the modern conception of the nodule as a nor- 
mal growth on the legume plant was established by Ij. C. Treviranus.^ 

Various theories have been proposed as to the function of these 
peculiar outgrowths, some advancing the idea that they were storage 
reservoirs or stimuli whereby the plants obtained nitrogen from the 
atmosphere thru their leaves. Recently Jost'* has called them "bac- 
terium galls," local hypertrophies not dissimilar to those sometimes 
caused by animal life. An astonishing conception has crept into the 
minds of the authors of certain general textbooks on bacteriology and 
plant physiology that nodules are abnormal growths and that their re- 
lationship to plants is either wholly or partially parasitic. It seems 
preferable, even to those familiar with the limitations of the theory, 
to describe this relationship as a normal condition and a true mutual 
symbiosis. 

That the formation of these nodules is due to external infection 
was definitely shown in 1887 by Marshall Ward,^ who was able to inocu- 
late the roots of young legumes by placing them in contact with old 
nodules. In Germany the first attempts to grow soybeans (Glycine 
Jmpida) in the botanical gardens resulted in failure, and it was not 
until soil from the natural habitat of that plant was imported for 
inoculation that soybeans were grown successfully.^ The history of 
the introduction of alfalfa culture in the states of Kansas and Illinois 
exemplifies in a large way this need of inoculation. From this experi- 
ence developed fhe soil-transfer method and tJie glue metliod'^ of inocu- 
lation, hotJi of ivliicJi are recognized today as superior to tlie use of so- 
colled commercial cultures. 

'Nodules are recognized on the following non-leguminous plants: alders 
(Alnus glutinosa), silverberry (Eleagnus), sweet gale {Myrica Gale), sago palm, 
an evergreen, (Podocarpineae) , cycads (Cycacadeae), birthwort (Arisiolociaceae) . 
Nitrogen-fixing bacteria resembling B. radicicola have been found in the alder, 
silverberry, sweet gale, and. five varieties of podocarpus. 

=Malphighi: Op. (1687), 2, 126, Leiden. 

'Treviranus: Bot. Ztg. (1853), 11, 393. 

.*Jost: Plant Physiology (Gibson 1907), 237. 

"Ward: Phil. Trans. Roy. Soc. London (1S87), 178, 139. 

'Soil inoculation experiments were instituted as early as 1887 at the Moor 
Culture Experiment Station, Bremen, Germany. 

'111. Agr. Exp. Sta. Buls. 76, 94. 



476 



Bulletin No. 179 



[March, 



Two types of nodules have been recognized by Tschirch;i Lupinus 
(lupine) represents one type and Robina (locust) the other. As may 
be seen by reference to Plates I and II, they differ in morphological 
appearance. The Lupinus tj^pe involves a swelling of the central root 
cylinders themselves, while in the Robina type only the epidermal and 
the endodermal tissues seem to enlarge. According to Tschirch, the 
nodules of lupines alone are of the first type, while those of all other 
legumes belong to the second. 

The figures in Plate III are sufficient to illustrate the most com- 
mon shapes of the Robina type. The shape varies with the different 
species of legumes, and to a certain extent with the individuals on the 
same legume plant. In the experimental work reported in this publi- 
cation, over twenty thousand nodules were examined closely, and it 
was not uncommon to find on the same plant notable variations due to 
external obstructions to growth. 

Infection 

The artificial inoculation of a plant is easily accomplished by con- 
tact. If the epidermis of the root is wounded and the infecting or- 
ganism {B. radicicola) brought into contact with the wound, nodules 





Fig. 1. — Boot hair of com- 
mon pea (Pisum sati- 
vum), showing infecting 
strand (x300) (After 
Prazmowski) 



Fig. 2. — Eoot cell, showing in- 
fecting strand passing thru it 
and the formation of lamellae 
(x650) (After Prazmowski) 



"Tschirch: Ber. deut. Bot. Gesell. (1887), 5, 58. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 



477 




Plate I,— Nodules op Eobina Type on Roots of Soybeans 
(Enlarged) 



478 



Bulletin No. 179 



[March, 




Plate II.— Nodules of Lupinus Type on Boots of Lupine Seedlings 
(After A. Meyer) 



1915] A Biochemical Study op Nitrogen in Certain Legumes 



479 




480 



Bulletin No. 179 



[March, 



result. Inoculation in pot cultures is attained by placing an infusion 
on the seed or in tlie medium. A similar method is successful with 
water cultures. 



Inoculation as It Occurs Under Field Conditions 

Studies of inoculation as it occurs in the field show the following 
generally accepted phenomena : 

As the tip of the root hair of the legume pushes itself out into 
the soil, it chances to come into intimate contact with the organism 
B. radicicola. Some scientists have exploited the view that the organ- 
ism is attracted to the plant by chemotaxis, believing that the plant 
excretes a substance, probably a carbohydrate, which diffuses into the 
soil solution and attracts the motile organism. While it has been 
rather definitely shown that this organism progresses in the soil at a 

rapid rate, nevertheless the 
number of root hairs in- 
fected^ is too small to lend 
support to a chemotactic 
theory. However the case 
may be, the organisms clus- 
ter at the tip of the hair 
and by means of an enzyme 
(or otherwise) rapidly dis- 
solve the cellulose of the 
cell wall, thus enabling the 
organism to enter the root 
hair. As a result, there is 
a decided bending of the 
tip, causing it to resemble 
a shepherd's crook. This 
was early observed as a 
sign of complete infection. 
It is claimed that other 
root hairs which form after 
infection are immune to 
the attack of other legumi- 
nous bacteria.2 

The organisms, by rapid 
division and growth, ad- 
vance thru the center of 




Fig. 3. — Young nodule magnified, showing af- 
fected root hair and same root hair more 
highly magnified (After Atkinson) 



the infected root hair. Prazmowski^ found organisms in the cell 

^Pierce, G. J.: Proc. Cal. Acad. Sci. II, No. 10 (1902), 295-328. Pierce found 
the proportion with bur clover to be 1 : 1000. 

'Fred: Vir. Agr. Exp. Sta. Ann. Ept. 1909-10, 123-125. 
^Prazmowski: Landw. Vers. Stat. (1890), 37, 160-238. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 481 

sap and even in the epidermis only two days after inoculation. 
In this advance an infection strand (Infektion-schlauche) is formed, 
which consists of gelatinous material, and in the earlier stages of de- 
velopment this strand may be traced from the root hair into the inner 
tissue of the root and from cell to cell thruout the nodule. This infect- 
ing strand is not supposed to constitute a portion of the living tissue, 
nor is it a well-defined tube; but, as Fred has recently shown, it con- 
sists of a large number of zoogloea occurring adjacent to one another, 
in which separate bacteria can be distinguished. The infecting strand 
branches profusely, and it was this habit of growth which caused the 
early investigators to consider it the mycelium of a fungous growth. 

Growth of the Nodule 

The presence of B. radicicola in the tissues of the root causes a 
rapid cell division in the pericycle. These cells become larger and 
contain more protoplasm than the surrounding cells, and as growth 
takes place, the cortical parenchyma and epidermis are forced out- 
ward, thus forming a nodule. The growth of the nodule is apical. The 
various tissues common to the plant are present (see Fig. 4). In the 
central portion of the nodule is the so-called bacteroidal tissue, which 
is ochre, flesh, or gray in color, according to the age of the nodule, and 
in this portion the infecting strand (Infektion-schlauche) is distin- 
guished in the young nodule. It ramifies thruout the cells, causing 
those which it enters to lose their power of cell division but not of 
growth. Later, or in older nodules, the infecting strand is not visible, 
and the bacteroidal tissue loses its firmness. At the period when seed 
formation is at its height, most of the nodules are soft, and the inter- 
nal tissues slough off, leaving the more resistant epidermal tissue as a 
mere shell, which later decays. The endurance of the nodule depends 
upon several factors, — chiefly, however, upon the kind of legume plant 
on which it is produced and the need of nitrogen by that plant. 

Pierce^ considers the nodules as originating endogcnously from 
the same layer of cells as the lateral roots, and as being morphologi- 
cally similar to them ; however, as the lateral roots rupture the epider- 
mis, the above statement is not entirely in accord with what actually 
takes place. 

The nodules are largest and most numerous where aeration is best 
in the soil. In saturated soils they occur at the surface and are often 
found colored green, very similar to sunburned potatoes. Nodules 
form in solutions, and exceptionally well in certain nutrient solutions. 
Several interesting instances have been brought to the attention of the 
Experiment Station, in which the observers believed that the nodules 

^Pierce, G. J.: Proc. Cal. Acad. Sci. 11, No. 10 (1902), 295-328. 



482 



Bulletin No. 179 



[March, 



had grown above the ground. These peculiarities were undoubtedly 
caused by unobserved physical conditions occurring at the time of in- 
fection or afterward. 



Jr^iicfir^J sTr 



o-r<^' 




auTt 



,0T 



rf-^ 



Tner'5' 



L^7-)clocJerrr)t<^ 



Fig. 4. — Young Nodule, Showing the Beginning of the Differentiation 
OF ITS Tissues (After Prazmowski) 

BACTERIOLOGICAL 

Minute bodies were first detected in nodules in 1866 by Woronin/ 
a Russian botanist. At that time bacteria were not recognized, and it 
was not until 1887 that they were demonstrated to be true bacteria by 
Wigand.^ 

'Woronin: Bot. Ztg. (1866), 24, 329. 

'Wigand: Bot. Heft. Forsch, a.d.Bot. Bart. 3 Marburg (1887), 288. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



483 



Immediately afterward, Beyerinck^ isolated the organism on an 
artificial medium composed of a decoction of pea leaves, gelatine (7 
percent), asparagine (.25 percent), and saccharose (.5 percent). He 
named the organism Bacillus radicicola, altho he described an organ- 
ism bearing a single polar flagellum. This organism became generally 
described as Pseudomonas radicicola, and some writers still prefer this 
designation. The organism has been known under a variety of terms, 
as ScJiinzia leguminosarum, CladocJiitrium tuberculorum, RJiizohium 
radicicola, RJiizohium leguminosarum. Bacterium radicicola. Micro- 
coccus tuherigenus, Myxobacteriaceae, Actinomyces, and Phytomoyxa. 
Such inappropriate names as RMzohacterium japonicum and Rliizo- 
hium sphaeroides are applied to certain special races. In commercial 
and general use, the organism is labeled as pea bacteria, bean bacteria, 
alfalfa bacteria, et cetera. 

Recent studies on the number of flagella possessed by this organ- 
ism have indicated that the organism is a bacillus, and it is there- 
fore desirable to adopt the original name B. radicicola, as proposed 
by Beyerinck. 

Bacillus radicicola 

These bacilli are rod-shaped organisms possessing numerous fla- 
gella (6 to 20), wldch are peritrichous. When full grown they vary in 
length from 1 to 4 or 5/*.^ It is not uncommon to find them from .5 to 
.6/A wide and from 2 to 3ju, long, and some have been found to measure 
only .18jLi wide and .9/^ long. The organism is actively motile. It is 

strongly aerobic, and in this connection 
Pierce calls attention to the intracellu- 
lar spaces in the root, which make it un- 
necessary to assume, as has been done, 
that it must live anaerobically. It is 
known that this organism does not form 
spores, but its means of enduring in the 
soil has not yet been determined. The 
bacilli prevail in the young nodule, 
while the branched forais, or bacteroids 
(see page 487), predominate in the older 
structure. 

B. radicicola grows well on a great va- 
riety of culture media, perhaps best on 
a medium of ash-maltose-agar or one of legume extract plus a sugar 
and dipotassium phosphate. Dextrose, saccharose, and maltose are 
suitable carbohydrates. The cultural characteristics of the colonies 
and the morphology of the organism will not be considered at this 
time, but it might be stated that many modifications occur on various 
media. 

'Beyerinck: Bot. Ztg. (1888), 46, 725; also 741, 757, 780, 797. 
*M=Diieron, or 1/25,000 of an inch. 




Fig. 5. — B. radicicola, show- 
ing shape and flagella 



484 Bulletin No. 179 [March, 

Growth and Endurance of B. radicicola 

The optimum temperature for B. radicicola varies between 18° 
and 26 °C. The thermal death point, according to Zipfel/ is 60° to 62° 
C. Growth is perceptible between 3° and 46° C. 

B. radicicola is not very sensitive to the reaction of the medium, 
which may be either acid or alkaline. Under field conditions, the 
organism exists in extremely acid soils, especially the race peculiar to 
legumes which thrive well on very acid soils. Experiments have dem- 
onstrated that the bacteria can withstand any degree of acidity or 
of alkalinity in the soil, that the particular legume itself can endure. 

That the organism endures at least two years in dry soil was de- 
termined by Ball.2 Harrison and Barlow^ found that the limit of 
viability on ash-maltose-agar varied somewhat, but that in the ma- 
jority of cases it was about two years. No doubt the organism will 
live much longer than this on artificial media when suitable conditions 
of growth are maintained. How long the clover or alfalfa organism 
will exist in a soil under field conditions is not yet known, but prac- 
tical observations indicate that it must be many years. 

The statement is quite generally made that B. radicicola becomes 
' ' nitrogen hungry ' ' when cultivated thru several generations on nitro- 
gen-free media. This fact has not been sufficiently demonstrated to 
be accepted, for while Siichtung, Hiltner, and others have found that 
these organisms survive cultivation on nitrogen-free media for a year 
and at the end of that time possess the same ability to effect inocula- 
tion and nitrogen fixation in the legume as organisms obtained from 
fresh nodules, yet the bacteria had apparently made no appreciable 
gain in ability to effect inoculation. Garman and Didlake^ failed to 
find that nitrogen-free medium possessed any particular advantage 
over a legume-extract medium in causing the organism to become 
' ' nitrogen hungry. ' ' 

Indentity op B. radicicola 

It is believed by some that the various legumes have different 
species of bacteria. Evidence has been produced which indicates 
that nodules do not contain but a single race of infecting organisms. 
Gnio de Eossi^ reported the finding, in artificial cultures, of two 
organisms which differed in that one formed a large hyaline colony, 
not developing well in beef and peptone gelatine, while the other 



^Zipfel: Centbl. f. Bakt. 2 Abt. (1912), 32, 97-137. Five minutes taken as 
the time of exposure instead of ten minutes. 

"Ball: Centbl. f. Bakt. 2 Abt. (1909), 23, 50. 

^Harrison and Barlow: Centbl. f. Bakt. 2 Abt. (1907), 19, 429. 

'Garman and Didlake: Ky. Agr. Exp. Sta. Bui. 184, 352. 

"^Gino de Eossi: Centbl. f. Bakt. (1907), 18, 289-314, 418-489. 



1915] A Biochemical Study op Nitrogen in Certain Legumes 485 

formed white non-transparent colonies in beef gelatine. He believed 
that he had found another organism associated with B. radicicola. 
This work has not been sufficiently substantiated to be accepted as 
final. Greig-Smith^ reported having found three races of this or- 
ganism in the same nodule. Hiltner and Stormer^ classify nodule 
bacteria into two groups, RJiizohium radicicola and RJiizohium heyer- 
inckii. The former they associate with lupines, serradella, and soy- 
beans ; the latter with all other legumes. 

On the other hand, the results of many investigators^ (especially 
Laurent,^ who obtained nodules on the pea with organisms from 
thirty-six different legumes, and Nobbe et al.,^ who worked on the 
adaptability of nodule bacteria of unlike origin in different genera of 
Leguminosae), seem to support the theories of the identity of nodule 
organisms and the presence of only one race in the nodule. 

On the whole, present experimental evidence is slightly in favor 
of the view that there is only one species of this organism thruout the 
entire family of legumes.*^ This conception is not easily reconciled 
with field observations, for under natural conditions this organism 
has become so modified as to make it appear that there are many 
species. Contamination of the nodule has undoubtedly been responsi- 
ble for varying conclusions in this connection. 

Enzyme Production by B. radicicola 

Hiltner'^ reported the finding, by filtration thru porcelain, of a 
substance produced by B. radicicola which can dissolve the cell wall 
of root hairs. No proteolytic enzyme has as yet been reported. 
Further, Beyerinck claims that no enzyme has been found which at- 
tacks lime, starch, or cellulose, or which is capable of inverting sac- 
charose. In recent studies, Fred,^ altho unable to detect a proteolytic 
enzyme, obtained slight evidence of the presence of oxidases in the 
slime of various legume bacteria. These results suggest the need of 
further studies on enzj^me production by this organism. Similar to 
all microorganisms, it has the ability to reduce methylene blue to the 
colorless leuco-compound. 



^Greig-Smith: Jour. Soc. Chem. Indus. (1902), 26, 304-306. 

^Hiltner and Stormer: Arb. K. Gsndhtsamt, Biol. Abt. (1903), 3H. 3, 151. 

'Harrison and Barlow: Centbl. 2 Abt. (1907), 19, 429. 

Kellerman: Centbl. f. Bakt. 2 Abt. (1912), 34, 45. 

Buchanan: Centbl. f. Bakt. 2 Abt. (1909), 22, 371. 

^Laurent: Exp. Sta. Rec. (1890), 2, 186. 

"Nobbe et al.: Centbl. f. Bakt. 2 Abt. (1895), 1, 199. 
" " '' : Ibid (1900), 6, 449-457. 

'Kellerman (see note 3) reported inoculation of soybean, lupine, and also 
of alfalfa from a culture originally isolated from alfalfa and kept on artificial 
media in the laboratory for six years. 

'Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 273. 
•Fred: Vir. Agr. Exp. Sta. Ann. Rpt. 1910-11, 123. 



486 Bulletin No. 179 [March, 

Slime Production by B. radicicola 

B. radicicola produces, on artificial media, a gum, or slime, which 
is partly soluble and party exists as a zoogloeal mass. The organisms, 
on suitable media, have been observed to surround themselves with 
definite capsules several times thicker than themselves. These cap- 
sules are rather distinct at first b\it later form a gelatinous mass. 
Greig-Smith^ and Maze,^ who studied this slime, claimed for it a nitro- 
genous substance. The results obtained by Buchanan, Gage, and 
Fred agree and are in direct refutation of the above. This is impor- 
tant to bear in mind in connection with the theories later to be dis- 
cussed. Gum is not formed from a carbohydrate containing less than 
five carbon atoms. 

Isolation of B, radicicola from Soil 

Until recently B. radicicola had never been very successfully 
isolated from a normal soil except by means of a legume plant. Gage,^ 
after a long, tedious process, obtained from soil an organism which 
was capable of producing nodules on red clover and which appeared 
to be identical with B. radicicola. Still more recently Greig-Smith^ 
has reported the isolation of this organism from soil, but his work 
has not been substantiated by others. 

Maze early attempted the isolation of B. radicicola from sterilized 
and non-sterilized soils to which pure cultures had been added. He 
isolated the organism from the soil which had been sterilized before 
the addition of the culture, but he was unable to recover it from the 
unsterilized soil. Kellerman and Leonard^ isolated (on the agar rec- 
ommended by Greig-Smith) an organism which inoculated alfalfa 
from soil that had been sterilized and subsequently inoculated with 
living organisms of B. radicicola. Lipman and Fowler*^ were able 
to isolate the organism peculiar to vetch {Vicia sicida) on soil-ex- 
tract agar and proved out the organism. They attained success in 
about 40 percent of the cases, judging from the condensed report 
recently published. 

Dissemination op B. radicicola 

Those familiar with pot-culture experiments and inoculation ex- 
periments with legumes easily understand that legume bacteria are 
disseminated in many ways. In fact, sterile conditions are difficult to 
maintain. The layman, however, may wonder how legume bacteria 

'Greig-Smith: Centbl. f. Bakt. 2 Abt. (1911), 30, 552-556. 
=Maze: Ann. Inst. Pasteur (1898), 12, 128. 
'Gage: Centbl. f. Bakt. 2 Abt. (1910), 27, 7-48. 
*Greig-Smith: Centbl. f. Bakt. 2 Abt. (1912), 34, 227-229. 
'Kellerman and Leonard: Science (1913), 38, 95-98. 
'Lipman and Fowler: Science (1915), 41, No. 1050, 256-258. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 487 

not common to a certain locality creep in. A few agents concerned in 
the transfer of the organisms are here cited. 

The seeds themselves are a common means of distribution. The 
ruptured seed coats offer an opportunity for the various kinds of bac- 
teria to accompany the seeds in a most persistent manner. Sometimes 
wind is responsible for the dissemination of these bacteria. An inter- 
esting instance is cited by Ball of Texas: a wind storm blew off the 
roof of the culture-house in which legumes were being grown under 
sterile conditions, and as a consequence the various plants under ob- 
servation became inoculated. Water has been known to aid in inocu- 
lating large areas during washing and floods. The transfer of un- 
cleaned seed is sure to result in the conveyance of some inoculating or- 
ganisms in the impurities accompanying the seed. Cultivation, espe- 
cially harrowing, is also responsible for the spread of the organisms. 
The addition to soils of legume residues from either fields or stables 
is still another common means of dissemination. 

Fixation of Nitrogen Without the Legume Plant 

Maze,i in 1897, demonstrated that nodule bacteria have the power 
to assimilate atmospheric nitrogen in the absence of a legume. His 
researches have been verified by others, altho the amounts of nitrogen 
obtained in his experiments have never been equaled. Recently con- 
ducted experiments^ on this question show that as an average, in liquid 
and in solid media, about 1.2 milligrams of nitrogen are fixed per 
100 cc. of medium. A fixation in the absence of the legume plant has 
been found in sterile sand and in soil. How important this kind of 
non-symbiotic fixation is, has yet to be more fully determined. At 
the present time it is generally recognized as insignificant compared 
with symbiotic fixation in the nodules of legumes. 

Bacteroids 

Bacteroids are believed to be a form which appears in the devel- 
opment of B. radicicola. The cell activities of this form are main- 
tained in a way similar to that in which the cell activities of the rod- 
shaped form are maintained. Stefan^ states that these bacteroids are 
thin-walled and capable of division when young, but that when older 
they become swollen and finally degenerate. Fred was able to observe 
the changes which occur in the organism in passing from the bacillus 
to the extreme vacuolized bacteroidal form. The organism at first 
apparently thickens at one end and then branches into the bacteroid, 
which is characterized by rounded outgrowths, kno^vn as vacuoles. 
These vacuoles appear at a definite period of growth and evidently 

'Maze: Ann. Inst. Pasteur (1897), 11, 44. 

'Fred: Vir. Agr. Exp. Sta. Ann. Ept. 1909-10, 138-142. 

'Stefan: Centbl. f. Bakt. 2 Abt. (1906), 16, 131-149. 



488 Bulletin No. 179 [March, 

are not a sign of polymorphism, but are a further development of the 
bacteroid. (They require special staining to be made visible.) 

^ 0^ Bacteroids occur in the nodule as 

<^ ,g^ esM <^ ^r" ^^ ^^ ^^ culture media. Their 

" morphology varies according to the 

^ ^ rm^ ^^ constituents of the culture media. 

Aj Some writers prefer to call these ir- 

tjj % <^ P P ^ Cl regular organisms degenerate or in- 

^^^ ^ volution forms. They were first ob- 

Fig 6.— Bacteroids, showing ^^^^^^ -^^ artificial media in 1888 by 

shape, and occurrence or • ^ . ^ ^ ^ t-,i 

vacuoles Eeyermck,! and have been studied by 

Hiltner,2 Stutzer,^ Buchanan,'* Fred,^"" 
and others. A medium rich 'in carbohydrates or the glucosides of 
amygdalin or salicin offers very favorable conditions for bacteroid 
formation. Of fifteen carbohydrates tested, mannite has proved par- 
.ticularly suited to their development. Glycerine is better than most 
nutrients, while the salts of organic acids have been found unsuitable. 

On careful observation the following factors have been found to 
exercise no influence upon bacteroid formation: temperature, light, 
osmotic pressure, decreased oxygen pressure, reaction of medium, ni- 
trogen-hunger, specific formative materials in the legumes, and the ac- 
cumulation of metabolic products. From the above observation it is 
evident that nutrition is a strong factor in bacteroid formation. 

It is claimed that each of the various legumes exhibits a different 
shaped bacteroid which is characteristic of that legame. In studies 
conducted at the Virginia Experiment Station the bacteroids pos- 
sessed by the Egyptian, the crimson, and the red clover were found to 
be very similar, while those of the vetch differed somewhat. More 
extended research regarding the appearance of bacteroids as connected 
with the beginning of nitrogen assimilation, is sorely needed. 

THEORIES OF ASSIMILATION, FIXATION, AND IMMUNITY 

Theories of Assimilation by the Plant 

In brief it may be said that the two main suppositions regarding 
assimilation are as follows: (1) that the bacteroids are bodily absorbed 
by the plant fluids; and (2) that the bacteroids, by some sort of 
change, produce the substance containing the assimilable nitrogen 
which the plant utilizes. 

The theory that the plant absorbs these bacteroids has been chal- 
lenged by some on the evidence which Nobbe and Hiltner*' produced 



^Beyerinck: Bot. Ztg. (1888), 46, 725. 

^'Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 273. 

'Stutzer: Ibid (1901), 7, 897. 

'Buchanan: Ibid. (1909), 23, 59-91. 

"Fred: Vir. Exp. Sta. Ann. Rpt. 1909-10, 128-198. 

"Nobbe and Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 449. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 489 

to show that a plant had fixed 1 gram of nitrogen while its nodules 
weighed only .3 gram. The relationship between the amount of nitro- 
gen fixed and the weight of the nodules is no criterion, however, for 
criticism of such a theory, inasmuch as this relationship is not at all 
definite but varies according to the development and needs of the 
plant. The failure to establish the presence of a proteolytic enzyme 
has also been responsible for no little criticism of the first supposition. 
While the second supposition seems more plausible, it must be ad- 
mitted that it, too, is only a theory which should be studied with the 
hope of isolating and identifying the diffusible substance. In connec- 
tion with this theory Golding^ conducted some very interesting experi- 
ments on the removal of the products of growth in the assimilation of 
nitrogen by legume bacteria. He reasoned that the plant played an 
important role in the removal of the products produced by bacteria 
in the nodule aside from the mere furnishing of suitable food. In his 
experiments he used a porous Chamberland filter-candle placed in a 
culture vessel to serve to imitate natural conditions. Aerobic condi- 
tions were obtained by passing purified air thru the cultures. The 
parts of the plants used in some of his experiments were sterilized in 
order to avoid the possibility of plant enzyme action. As a result of 
his method of experimentation, he obtained a much greater fixation of 
nitrogen than other experimenters had found, and the logical conclu- 
sion arose that the plant performs a function in the assimilation 
of nitrogen which is construed to be the removal of soluble products 
of growth. The results of Golding's most extensive experiment are 

embodied in the table below : 

N. in 
grams 

500.0 grams of Stems and Leaves 2.86.3 

20.2 grams of Eoots and Nodules (quite frei h) 094 

3000.0 cc. Ammonia-free Distilled water 000 

Total Nitrogen to start with 2.959 

2870.0 cc. Filtrates and Drainings 731 

566.2 grams of Wet Res^idue 2.570 

To'al Nitrogen after experiment 3.301 

Total Gain of Nitrogen during experiment 342 

Theories Regarding the Chemical Phenomena of Fixation 

As yet, purely chemical theories of fixation arc entirely hypo- 
thetical ; however, they deserve consideration, for even theories un- 
supported by facts may have a value in stimulating thought and as- 
sisting in the development of more rational views. 

"Frank,2 a prominent Frenchman, was among the first to attempt 

•Gelding: Jour. Agr. Sci. (1905), 1, 59-04. 

Trank: l>andw. Jalirl). (1888), 17, 504-518; 19, 564. 



490 Bulletin No. 179 [March, 

an explanation of how the plant actually obtains nitrogen. He be- 
lieved that it came in thru the leaves, and even recently some isolated 
statements hold to this idea. His view was allied with the conception 
of stimulation which many held ; namely, that the organisms on the 
root stimulated the plant to fix nitrogen in its leaves. Stocklasa,^ as 
a result of his chemical investigations, also believed that assimilation 
took place thru the leaves, — that amides were first formed, and that 
these, migrating to the nodules, reacted with glucose and produced 
protein, which served as the nutrient medium for the bacteria. In 
this connection he advanced the idea that the bacteria produced an 
enzyme which enabled the plant to effect this fixation. 

Loew and Aso^ in 1908 suggested that ammonium nitrite was the 
first compound produced, the nitrous acid being readily reduced to 
ammonia. Little evidence has been ol)tained to suj)port this theory ; 
no evidence whatever has been found under controlled conditions. 

Gautier and Drouin^ suggested that the nitrogen is oxidized to 
nitric and nitrous acid. Winogradsky^ has advanced the idea that the 
free nitrogen in the plasma of the organism may unite with nascent 
hydrogen and form ammonia, which by oxidation would become assimi- 
lable. In connection with the two latter theories, it should be empha- 
sized that the presence of nitrites, nitrates, or ammonia in the nodules, 
roots, or tops of legumes, inoculated or uninoculated, when grown in 
the entire absence of combined nitrogen, has not been established. 

Gerlach and VogeP have investigated non-symbiotic nitrogen 
fixation and have arrived at the conclusion that there is a direct union 
of free nitrogen with some organic compound inside the bacterial cell. 
Heinze*' thinks it probable that nitrogen is at once brought into com- 
bination with a hydrocarbon (glycogen), and suggests that a salt of 
carbamic acid maj^ be formed first or that carbamic acid may be pro- 
duced from cyanamid. 

There is yet another most extraordinary theory which, owing to 
its somewhat recent notoriety, it seems appropriate to consider. This 
theory is most properly called the Jamicson theory. The rather pecu- 
liar views embodied in it are perhaps quite well explained in an article 
published in TJie Spokesman Review, Spokane, Washington, March 28, 
1913. Tlie Review is a bi-weekly paper devoted to agricultural inter- 
ests. 



^Stocklasa: Laiidw. Jalirb. (1895), 24, 827-863. 
=Loew and Aso: Bui. Col. Agr. Tokyo Imp. Univ. (1908), 7, 507. 
^Gautier and Drouin: Bui. See. Chim. Paris 78, 84-97. 
'Winogradsky: Centbl. f. Bakt. 2 Abt. (1901), 7, 842. 

'Gerlach and Vogel: Centbl. f. Bakt. 2 Abt. (1902), 9, 817-821, 881-892; 
(1903) 10, 636-644. 

''Heinze: Landw. Jahrb. (1906), 35, 907. 



1!)15] A Biochemical Study op Nitrogen in Certain Legumes 491 

"Do Plants Directly Absorb Free Nitrogen from the Air? 
' ' Scotch Scientist is at Odds with the Common Belief 

"The doctrine that plants directly absorb free nitrogen from the air con- 
flicts with the earlier beliefs. It is held that plants, with the exception of 
legumes, cannot utilize the nitrogen of the air, the explanation in the eaee of 
legumes being that by the aid of nitrogen in organisms on their roots the?e 
plants utilize atmospheric nitrogen. 

' ' Thomas Jamieson, Director of the Agi-icultural Eesearch Association of 
Scotland, takes issue with this belief. Mr. Jamieson has made a life study of 
the problems of plant nutrition. An abstract of his views is given in the New 
Zealand Journal of Agriculture. Mr. Jamieson proceeds to show: 

' ' 1. That the legume-tubercular theory is untenable. 

' ' 2. That the nitrogen of the air is directly used. 

"3. That the application of this knowledge is valuable to the agricul- 
turalist **********. 

' ' Mr. Jamieson disagrees with the theory that the tubercle formation on 
leguminous plants is a normal growth containing a net structure of plant food 
thru the union of fungus and a legume. He says : 

" '1 regard the tubercles as abnormal growths. I hold that no "symbiotic" 
action takes place ; that the fungus is not a fixer of the nitrogen ; that the 
legume plant is itself the fixer, and that it fends its manufactured albuminous 
products to heal up the wound or to counteract the drain of the parasitic fungus ; 
and that the tubercle has nothing to do with the fixation of the nitrogen of 
the air ****************' 

"As to~ the explanation of tubercles of leguminous plants Mr. Jamieson 
says: 

" 'The plant being attacked by the fungus, a wound is made, the fluid of 
the plant courses to repair it, and not only is the leguminous fluid of the plant 
rich in nitrogen, but its most nitrogenous fluid, albumen, is just a plastic material 
like the white of an egg, especially suited to heal the wound and to form a sac 
round the invader. 

" 'There is nothing exceptional in the bearing of tubercles by the legume. 
The nodules, or tubercles, are well displayed. The legume is a plant specially 
sought by fungus demanding nitrogen. It is provided with a means of supplying 
the element, hence it is specially attacked. ' 

"Further investigations lead Mr. Jamieson to the conclusion that nature pro- 
vides special means for all plants to absorb nitrogen. Even the hardest leaves 
are soft in the earlier stage. The cultivated members of the legume family have 
broad, soft leaves studded with apertures, supposed to serve for exhalation. It 
is accepted that the green cells or the chlorophyl contained by these cells de- 
compose the carbonic-acid gas. Cannot a similar action extend to nitrogen? 



**#»****«»»****^ 



"The effect on the soil of producing certain crops, as cereals and grasses, 
is to reduce the available plant foods. Of these, in their simple forms, the most 
important supplied by fertilizers are nitrogen, phosphorus, and potash, and of 
these the farmer can avoid the expense of the purchase of nitrogenous manures 
by the adoption of a rotation to include those plants that are rich in nitrogen. 
This is not new. Legumes have been availed of from the earliest recorded time 
as preparatory to cereals. What is new is that there is a wider field of plants 
for selection and the farmer knows why these jdants enrich the soil for the ni- 
trogen-demanding cereals. The plants, among others mentioned by Mr. Jamieson, 
are rape, mustard, and turnips." 

The above theory has received more contradiction and less sup- 
port than the others reviewed. ^ 



'Henry: Ann. Sci. Agron. (1909), 26, 102-130. 
Vageler: Centbl. f. Bakt. 2 Abt. <1909), 22, 452. 
Kovessi: Compt. Eend. Acad. Sci. (1909), 149, 56. 
Kny: Ber. deut. Bot. Gesell. (1909), 27, 532. 
Mameli and Pollacci: Ann. Sci. Agron. (1914), 31, 141. 



492 Bulletin No. 179 [March, 

Three possible chemical processes in fixation have been consid- 
ered by scientists: 

1. Keduction 

2. Oxidation 

3. Direct union into an organic compound 

The first two possible processes have received no chemical verification, 
while the third is supported only by data which are of an eliminative 
character. More data of a similar nature will be found in Part II 
of the experimental section of this bulletin. 

It is interesting to note that opinion seems to be strengthening 
in support of the theory of the direct union of nitrogen gas into 
organic combination, in spite of the fact that such a combination is 
unknown in chemistry to take place at ordinary temperatures. It is 
possible, however, to unite nitrogen into organic combination at at- 
mospheric pressure, altho a high temperature is required. 

Theories op Immunity 

Reasoning from animal life, it seems logical for one to believe 
that the relative strength of a legume plant or of B. radicicola may 
vary under certain conditions so that the plant will resist the entrance 
of the organism. Inoculation experiments have produced data show- 
ing that B. radicicola causes a certain resistance on the part of the 
plant, making it necessary in some cases to employ organisms of 
greater efficiency in order to produce inoculation. 

Hiltner^ has given the six following conditions as instances in 
which immunity demonstrates itself. 

1. The organisms cannot get into the plant. 

2. The organisms gain admission into the plant but do not pro- 
duce nodules because the plant, by its greater resistance, absorbs the 
bacteria. 

3. The organisms enter the plant and produce nodules, but no 
fixation of nitrogen occurs. 

4. The organisms enter, produce nodules, and nitrogen is fixed 
and assimilated by the plant. 

5. The organisms are so efficient in comparison with the plant 
that the latter is injured. 

6. The organisms are parasitic and the plant is actually killed. 

In the pursuance of the investigations reported in this bulletin 
no indication of the existence of any of these conditions, except No. 
4, was observed, and in no instance under normal conditions did in- 
oculation fail to produce nodules and cause a fixation and an assimi- 
lation of nitrogen. 

^Lafar: Handbuch der technischen Mykologie (1904-6), 3, 45. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 493 

Various other similar theories of resistance have been proposed, all 
of which are permeated with the idea of natural or acquired immunity. 
Prominent among these might be mentioned Siichtung's theory of 
equilibrium.! Siichtung assumed that the bacteria produced a toxin 
and the plant an antitoxin, and that the degree of equilibrium deter- 
mined the extent of nodule formation, the plant becoming immunized 
by an antibody and not by a substance produced by the bacteria; 
further, that the nitrogen supply in the plant was regulated by the 
production of this antibody. It is conceivable that in some of the cases 
observed the apparent immunity may have been due to a weakness on 
the part of the bacteria rather than to resistance by the plant. Siich- 
tung's equilibrium theory considers varying conditions of virulence on 
the part of the bacteria and varying degrees of resisting ability by the 
plants. His theory was advanced as the result of carefully conducted 
experiments which showed that there were variations in virulence 
between organisms of the same kind when grown upon artificial media 
and when obtained from fresh nodules. 

The inoculation of legumes in solution is inhibited by potassium 
nitrate, tho a convincing explanation of this inhibition has not yet 
been offered. Some experimenters believe that the immunity of a 
plant is strengthened by its nitrogen nutrition ; others hold that 
bacteria find another source of nitrogen nutrition in the nitrate and 
hence do not seek the plant. It has been recently shown, however, 
that the organism will produce nodules after the concentration of the 
nitrate has been reduced by the plant, which would tend to show that 
the immunity of a plant is not strengthened and that the organism 
is not permanently injured by a solution of potassium nitrate suit- 
able for the plant. 

Inoculation under field conditions is no doubt inhibited by physi- 
cal and antagonistic biological factors, which have been considered 
only briefly by most investigators. 

The subject of immunity in plants has been given little attention 
thus far, but the increasing number of bacterial diseases in the plant 
kingdom will undoubtedly lead to research in this direction. It is well 
known that bacterial diseases of plants are the most difficult to control ; 
the need of investigation in this unexplored field of immunity as an 
aid in their control is imperative. 

PRACTICAL CONSIDERATIONS WITH REGARD TO 
LEGUME FIXATION 

Mutual Symbiosis 

-Mutual symbiosis may be defined as the contiguous association of 
two or more morphologically distinct organisms not of the same kind, 

'Suchtung: Centbl. f. Bakt. 2 Abt. (1904), 11, 377. 



494 Bulletin No. 179 [March, 

resulting in an acquisition of assimilated food substances. It implies 
that the organisms concerned have the power of independent exist- 
ence, but that both are benefited by the close association. 

The relationship existing between B. radicicola and legumes is 
one of mutual sjonbiosis. The facts which bear out this belief are too 
convincing to need explanation. However, some prefer to call the re- 
lationship a truly parasitic condition, while others consider it to be 
parasitic in the beginning and later a true mutual symbiosis. This 
latter conception would seem to be plausible, yet no exact data have 
been produced to show that a parasitic condition exists at any stage. ^ 

The result of this mutual symbiosis is wonderfully characteristic 
of nature as well as astounding when one considers the corresponding 
chemical process, in which the energy expended is so apparent and the 
temperature required so high.^ The energy values in the symbiotic 
fixation of nitrogen by B. radicicola and legumes have never been de- 
termined. "When B. radicicola and Azotohacter are grown under simi- 
lar conditions, apart from their respective hosts,^ less organic carbon 
per unit of nitrogen fixed is oxidized by B. radicicola than by Azoto- 
hacter. Present knowledge indicates that a very great amount of 
energy is necessary for the fixation of atmospheric nitrogen by Azoto- 
hacter. 

Table 1 presents the amounts of some of the common materials 
that must undergo rather complete oxidation in order to furnish suf- 
ficient energy for the addition of fifteen pounds of atmospheric nitro- 
gen to the surface soil of an acre by Azotohacier. The figures show 
that in the case of dextrose 66% times as much organic matter is re- 

Table 1. — Amounts of Materials Necessary for the Fixation of Fifteen 
Pounds of Atmospheric Nitrogen per Acre by Azotobacter 

Kind of material 



Dextrose (sugar) . . 
Fresh clover tops. 
Fresli lupine tops. 

Wheat straw 

Corn stover 

Oak leaves 



Pounds 


required 


1 


000^ 


1 


212 


2 


000 


4 


300 


5 


500 


11 


500 



'This figure represents the minimum amount of dextrose consumed per unit 
of nitrogen fixed; in other words, 1 gram of dextrose (yielding 3,750 calories) 
is necessary for the fixation of 15 milligrams of atmospheric nitrogen by Azoto- 
hacter. 



^Experiments are now in progress at this station with the view of obtaining 
data on this question. 

"The most recent figures show that 1 kilowatt hour yields 70 gi-ams of nitrogen 
in the form of cyanamid ; in other words, 1.35 horse-power yields 70 grams, or 
8.74 horse-power per hour yields 1 pound of nitrogen. 

*Algae are understood as the host for Azotohacter. The word host as used 
in this publication is not intended to convey the idea of parasitism. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 495 

quired as there is nitrogen fixed, and in the case of oak leaves, 766 
times as much. In the oxidation of such large amounts of organic 
carbon it is easily seen that the volume of organic matter in the soil is 
greatly reduced. 

It has been definitely shown that Azotohacter lives in a symbiotic 
relationship with algae. It is also well known that our normal soils 
possess an abundant algal flora. In view of these two facts it may 
possibly be found that nitrogen is accumulated by Azotohacter with- 
out the above reduction in the volume of the organic matter of the soil. 

Whatever the future may disclose, the only fact that now re- 
mains to be pointed out is that in the symbiosis between legumes and 
B. radicicola, instead of there being a decrease in the organic matter 
of a soil, a material increase is bound to result. While fixation pro- 
gresses, organic matter is being manufactured by the plant, which is 
later returned to the soil. This process is, then, a constructive one, as 
compared with the destructive non-symbiotic fixation. 

Amount op Nitrogen Fixed per Acre per Year 

The amount of nitrogen added to a soil depends in part upon the 
relative supply of that element in the soluble and decomposable forms, 
organic as well as inorganic. The poorer the soil the greater the 
amount of nitrogen that will be fixed, tho in a rich soil in which the 
nitrogen is not in an available form, large amounts may be fixed. 
For instance, altho a peat soil contains in one acre some thirty 
thousand pounds of nitrogen in the surf",ce million pounds (0 to 6% 
inches), yet because of a lack of proper organisms in the soil to 
decompose the organic matter, which resists natural decay and there- 
fore does- not readily furnish nitrogen to plants, a legume crop may 
add large amounts of that element. 

Where nitrates are present in large amounts, they are taken up 
by legumes; but where they are present in only small amounts, as is 
the case during dry seasons even on the common prairie corn-belt land, 
atmospheric nitrogen is fixed by legumes. The fixation varies with 
the seasonal conditions, a hot, moist season being best suited to the 
summer legumes. Among other factors the kind of legume and the 
duration of its growing period affect the amount of nitrogen added. 
The annual legumes must necessarily fix nitrogen much faster than 
the biennial or perennial legumes. The yield does not necessarily in- 
dicate the amount of fixation, as some legumes which yield much less 
hay and seed than others may have a greater total nitrogen content. 

The most reliable data which now exist indicate that two-thirds 
of the nitrogen in legumes grown on soils of normal productive power 
is obtained from the air.^ These figures, contributed by the Illinois 

'Hopkins: 111. Agr. Exp. Sta. Buls. 76 and 94. 



496 Bulletin No. 179 [March, 

Experiment Station, were obtained by analyzing inoculated and unin- 
oculated legumes from like areas of normal soils, and as a result of pot 
experiments. Computed by these data, a 3-ton crop of cowpea hay 
adds 86 pounds of nitrogen per acre, a 25-bushel crop of soybeans 
with 21/4 tons of straw adds 106 pounds, a 4-ton clover crop adds 106 
pounds, and a 4-ton alfalfa crop adds 132 pounds. 

A nitrogen gain of 200 pounds per acre has been reported by the 
New Jersey Experiment Station^ with crimson clover. At the Rhode 
Island Experiment Station," as a result of a pot-culture experiment, it 
was found that nitrogen had been added at the rate of 400 pounds per 
acre per year. This experiment extended over five years, and legumes 
were grown both in the summer and in the winter. The tops of the 
summer legumes (cowpeas and soybeans) were removed from the soil, 
while the winter legume (vetch) v»^as turned back into the soil. It 
should be noted, however, that an acre of this soil to a depth of 6% 
inches contained only a little over three thousand pounds of nitrogen. 
Moreover, in this experiment optimum conditions were established, 
and no losses were possible from drainage, — which factors would tend 
to make these results much higher than would be obtained under field 
conditions. 

In considering soil enrichment by clover, ten years' results of a 
field experiment at the Experimental Farms, Ottawa, Canada,^ are 
important. In this experiment a light, sandy loam with a sandy sub- 
soil was planted to clover continuously, being reseeded every two 
years. The clover was cut and left to decay on the land. In ten 
years the nitrogen content of this soil was doubled. The yearly gain 
of nitrogen was fifty pounds per acre. It was found that from two 
to three times that amount was added, but that all but fifty pounds 
was dissipated by bacterial activities and in other natural ways. 
Analyses of the clover crop also brought out the fact previously men- 
tioned that the amount of nitrogen fixed is influenced in part by the 
season. 

Value of Legumes as Nitrogen Retainers 

Legumes have a very great value aside from their role in the 
nitrogen-fixing process. It is well known that they require more 
nitrogen for their growth than other ordinary farm crops, and that 
they therefore contain more of it per ton. It seems very appropriate, 
therefore, to select legumes for such purposes as holding soil from 



'N. J. Agr. Exp. Sta. Ept. 1894, 158. 
-R. I. Agr. Exp. Sta. Bui. 152. 
^Experiiueutal Farms, Ottawa, Ept. 1912, 145. 



1015] A Biochemical Study of Nitrogen in Certain Legumes 497 

washing and preventing sands from shifting, for not only do they 
serve these purposes well, but at the same time they conserve rela- 
tively more nitrates from loss than non-legumes. Of course a con- 
dition might occur in which a legume would draw all its nitrogen 
from the soil, but even in such a case a legume would be preferable 
to a non-legume, as by its use relatively more nitrogen would be kept 
from leaching and so saved for future crops. 

Cross-Inoculation 

There are relatively few cases of cross-inoculation that have been 
definitely determined as occurring under natural conditions. The 
most important example is the cross-inoculation that takes place be- 
tween the sweet clovers and alfalfa. Bur clover (Medico go lupu- 
lina) is another source of inoculation for alfalfa. The wild vetches 
serve for inoculation of the cultivated vetches. It w^ould seem that 
many such cases may exist in which the wild specie of a legume con- 
tains the organism for the inoculation of the cultivated legume of the 
same specie or even of an entirely different specie or genera. Inves- 
tigations along this line have not been carefully undertaken as yet. 

It has been possible under laboratory conditions to cross-inocu- 
late in many different ways. The data furnished by Laurent, re- 
ferred to in an earlier part of this publication (page 485), together 
with that furnished l)y Moore, Kellerman and Leonard, and others, 
is of interest. Laurent produced nodules on the pea with the organ- 
isms from thirty-six different species of legumes. Moore^ produced 
nodules on many legumes with the pea organism, among which were 
crimson clover (Trifolium incarnatinn), white clover {Trifolium re- 
pens), red clover {Trifolium. pratense), berseem {Trifolium Alexan- 
drinum), alsike {Trifolium. lijihridium) , sweet clover {Melilotus alba), 
cowpea {Vigna catjang), alfalfa {Medicago .sofwa), broad bean {Vicia 
faba), common bean {PJiaseolus vidgnris), fenugreek {Trifolium foe- 
num graecum), hairy vetch {Vicia villosa) , scarlet vetch {Vicia ful- 
geus), and yellow vetch {Vicia lutea). The results published by 
Kellerman and Leonard represent the extreme in cross-inoculation at 
the present time. It will be recalled that they have reported the 
inoculation of the soybean, the lupine, and alfalfa with an organism 
originally obtained from alfalfa nodules,^ altho it has been quite 
generally believed that the soybean was representative of a special 
class as regards its inoculation, and the same can be said regarding 
the lupine. Legumes may be grouped as follows according as their 



^Moore: U. S. Dept. Agr. Bur. Plant Indus. Bui. 71. 
^See page 485, note 6. 



498 Bulletin No. 179 [March, 

bacteria are interchangeable for the purposes of inoculation: 

Group 1 Alfalfa, sweet clovers, bur clover, 

black niedick 
' ' 2 All true clovers 
' ' 3 Cowpea, partridge pea 
' ' 4 Soybean 
" 5 Bean 
" 6 Peas (garden and field), vetches (cultivated 

and wild), sweet peas, lentils 
' ' 7 Lupines 
' ' 8 Sanfoin 
' ' 9 Locust 

Nobbe, Hiltner, and SchmicP obtained inoculation and nitrogen 
fixation with the locust and vetch cross-inoculated with each other and 
with pea bacteria, as shown below : 





Inoculated with 


Locust 


Vetch 


Pea-bacteria 


Nitrogen 


Locust (Eobina) 


232.1 


13.5 


21.2 


assimilated 


Vetch (Vicia) 


12.9 


264.0 


22.6 



Some prefer to divide the legume bacteria into two classes accord- 
ing to the beneficial or detrimental effect produced by lime upon the 
legume. In this classification, alfalfa would represent one type and 
serradella the other. 

The question of cross-inoculation is far from settled. It is easily 
seen that a great many interesting problems, aside from the purely 
scientific studies of the laboratory, are presented for the soil biologist 
in the pursuance of this field of research. 

Associative Growth of Legumes and Non-Legumes 

The associative growth of legumes and non-legumes has been 
given renewed notoriety in recent publications. Practical observa- 
tions of long standing have indicated that a non-legume benefits by the 
presence of a legume during the second year of its growth — as might 
reasonably be anticipated. The proof of a benefit by association dur- 
ing the first season is not sufficiently established for a generalization, 
for errors in sampling, in methods of experimentation, and other un- 
favorable conditions have crept in and overshadowed the full value of 
the data reported. 

The problem of associative growth involves many details that 
must be further studied. The stimulation caused by the struggle for 
existence in association may increase the height of the crops or the 
amount of the organic matter produced, yet not necessarily the nitro- 
gen content. In the work under observation at this station, it appears 
that the nitrogen Avhich is returned to the soil as the nodule sloughs 
off: could hardly be utilized by an ordinary annual non-legume crop. 
It is yet to be determined whether either the legume itself or its 

^Nobbe, Hiltner, and Schmid: Landw. Vers. Stat. (1894), 45, 12. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



499 



nodules exude nitrogenous compounds during their active period of 
growth. 

CHEMICAL 

The chemical composition of legumes from the standpoint of their 
nitrogenous constituents has been investigated to some extent, but the 
studies closely related to this point are relatively few. The follow- 
ing data are very general in character and relate to studies concern- 
ing the total nitrogen content of the different parts of legumes at dif- 
ferent periods of growth. Studies upon some of the various nitroge- 
nous compounds are also included. 

In 1895 Stocklasa/ working with lupines {Lupinus luteus 
and Lupinus augusfifolius), found that the nodules were richest in 
the element nitrogen at the time of blooming, while the roots appeared 
to be richest in that element at the fruiting period. His results are 
given in Table 2. The figures for the nodules indicate that the nitro- 
gen is either taken up by the plant for seed production or diffused 
into the soil. 



Table 2. — Total Nitrogen in Lupinus Luteus: 
BY Stocklasa 



Results Obtained 





(Percentage on 


dry basis) 




Period 


Roots 


Nodules 


Blooming 

Fruiting 

Maturity 


1.64 
1.84 
1.42 


5.22 
2.61 
1.73 







Stocklasa also determined protein, amides, and aspai'agine in lu- 
pine nodules. The protein was obtained by the Statzer method, the 
amides by the Kjeldahl method, and the asparagine by calculation 
from the ammonia obtained by distillation with magnesium oxid. 
Table 3 shows his results. 



Table 3. — Nitrogen Compounds in Lupine Nodules: 

BY Stocklasa 



Results Obtained 





(Percentage on dry basis) 




Period 


Protein 


Amides 


Asparagine 


Blossoming 


3.99 
1.54 


.35 
.15 


.34 


Maturity 


Trace 



The presence of asparagine in the nodule is important, as it is 
thought to be intimately related with the formation of protein. 

In 1901 Wassilieff- studied the nitrogen compounds in white 
lupine {Lupinus alba) seeds and seedlings. He found that the seeds 
contained 7.G8 percent of total nitrogen; and that of this, 6.89 percent 
was in the form of protein and .53 percent was precipitated by phos- 

•Stocklasa: Landw. Jahrb. (1895), 24, 827-863. 
^'Wassilieff: Landw. Vers. Stat. (1901), 55, 45-77. 



500 



Bulletin No. 179 



[March, 



photungstic acid, leaving a difference of .26 percent, asparagine. The 
occurrence of asparagine in large amounts in the seedlings is shown 
b}' the data given in Table 4. 

Table 4. — Nitrogen Compounds in Pourteen-Day-Old Green Seedlings of 

White Lupines: Kesults Obtained by Wassiliefp 

(Expressed in percentage on dry basis) 



Parts 


P. T. A.^ 
nitrogen 


Asparagine 


Protein 


Total 
nitrogen 


Leaves 


.53 
.63 
.42 
.46 


1.45 
3.83 
4.57 
2.20 


4.11 

2.44 
1.56 

1.87 


6.57 


Cotyledons 

Stems 


7.83 
6.77 


Roots 


5.40 







^P.T.A. : This abbreviation for phosphotimgstic acid will be used thrnout 
this publication. 

Wassilieff also demonstrated the presence of leucine and tyrosine 
in the cotyledons of one-week-old seedlings of white lupines. These 
and other amino acids would be expected to be present when the pro- 
tein of the seed is breaking down for the nutrition of the seedling. 

Knisely^ analyzed the leaves, pods, stems, roots, and nodules of 
lupine plants for total nitrogen at three distinct periods of develop- 
ment. His results show better than the others presented where the 
nitrogen accumulates as the plant matures. 

Table 5. — Total Nitrogen in Lupines: Results Obtained by Knisely 
(Expressed in percentage on dry basis) 



Period 


Leaves 


Pods 

3.07 
3.38 
3.68 


Stems 


Roots 


Nodules 


Full bloom 

Pods well formed 

Pods very large 


4.02 
3.70 
3.41 


1.15 

.88 
.90 


.92 
.83 
.66 


5.17 
4.29 
3 70 







Schulze and Barbieri- examined lupine and soybeans seeds and 
seedlings for nitrogen and obtained the results shown ' in Table 6. 



Table 6. — Nitrogen in Lupine and Soybean Seeds and Seedlings: 
Obtained by Schulze and Barbieri 



Results 



(Express 


ed in percentage on dry basis) 




Material 


Total 
nitrogen 


Protein 


P.T.A. 

nitrogen 

.24 
.13 

1.60 

2.17 

.56 


Filtrates 
from P.T.A. 


Lupine seeds 


8.63 
6.73 

10.64 

10.51 

7.42 


8.17 
0.32 

3.40 

2.33 

3.86 


2^ 


Soybeans 


28 


Lupine dark seedlings 

11 to 12 days old 

Lupine dark seedlings 

12 days old 


5.64 
6 01 


Soybean seedlings 

15 days old 


3.00 



'Knisely: Ore. Agr. Exp. Sta. Rpt. 1909, 30-31. 

^Schulze and Barbieri: Landw. Vers. Stat. (1881), 26, 241. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



501 



They also found a large amount of asparagine in both the lupine 
and the soybean seedlings. 

Schulze^ has made a careful study of the compounds in plants, 
and has formulated the hypothesis that the same decomposition 
products arise from protein in the plant as outside it, but that in 
the plant the compounds are further altered, thereby affecting in 
varying degree the individual products of the hydrolytic decomposi- 
tion. A comparison of the analyses of pea seedlings one week old 
and those three weeks old showed the following dilt'erences : 



1 week. 
3 weeks. 



Leucine 


Tyrosine 


Arginine 


Asparagine 


abundant 


little 


present 


absent 


much less 


absent 


almost absent 


very abundant 



Arginine and amido acids w^ere shown to be present in the lupine 
cotyledons, but asparagine was absent, altho the latter substance was 
found in the stem of the seedling. It has been suggested that the oc- 
currence of asparagine is associated with the disappearance of amido 
acids and not of protein. Phenyl alanine, tyrosine, and tryptophane 
have been reported in the white lupine {Lupinus alha), tyrosine and 
tryptophane in vetch {Vicia sativa), and tryptophane in the garden 
pea {Pisuni sativum).- 

Smith and Robinson^ found 4.19 percent of nitrogen in soybean 
nodules and 3.90 percent in cowpea nodules. They observed that inocu- 
hition increased the protein content of soybean plants without in- 
creasing the yield of beans. This has been noted by other experi- 
menters. 

Hopkins^ has reported the analyses of cowpea plants for total 
nitrogen with and without inoculation. The nodules, roots, and tops 
were analyzed separately, as will be seen by reference to Table 7. 



Table 7.- — Nitrogen Fixation by Cowpeas: Kesults Obtained by Hopkins 

(Expressed in egs.) 



Treatment 


Tops 


Eoots 


Nodules 


Nitrogen 
fixed 




146 
38 

171 
55 

143 
40 


9 
3 
10 
4 
8 
4 


11 
18 
17 


125 


Ten plants without bacteria 


140 








124 













^Schulze: Zeits. f. Physiol. Chem. (1895), 24, 18; 30, 241. 
=Schulze et al: Zeits. f. Physiol. Chem. (1887), 11, 43; (1906), 48, 387, 396; 
(1910), 65, 431. 

Gorup Besamez: Ber. deut. Chem. Gesell. (1887), 10, 781. 
'Smith and Robinson: Mich. Agr. Exp. Sta. Bui. 224, 125-132. 
-Hopkins: 111. Agr. Exp. Sta. Bui. 94, 319. 



502 



Bulletin No. 179 



[March, 



The inoculated plants contained a much greater percentage of 
nitrogen than the uninoculated, the average content of the inoculated 
being 4.24 percent in the tops, 1.48 percent in the roots, and 5.92 per- 
cent in the nodules, while the average content of the uninoculated was 
2.48 percent in the tops and .88 percent in the roots. 

The ash and the ash constituents of the nodules and the roots of 
lupines have been determined by Stocklasa,^ as presented in Table 8. 
The total ash of the nodules was found to be 6.32 percent, while that 
of the roots was found to be 4.55 percent. 



Table 8. — Ash Constituents in Lupine Nodules and Roots; 
Results Obtained by Stocklasa 

(Expressed in percentage) 



Constituents 


Nodules 


Roots 


Si 


1.59 

4.90 

(5.51 

17.31 

16.94 

7.41 

7.64 

.83 


1.90 


S 

P 

K 

Na 

Me 


0.38 

4.28 

12.05 

19.94 

7.05 


Ca 

Fe 


12.04 
.75 



The analyses of red-clover nodules show a potassium content of 
2.63 percent in the dry matter.^ The nodules, therefore, are rela- 
tively rich in mineral elements as well as nitrogen compounds ; and 
Stocklasa 's results (see Table 8) show that the chief differences 
between the roots and the nodules in the composition of the ash 
constituents are in phosphorus, potassium, calcium, and sodium. 
The nodules are richer in the first two elements and the roots in the 
latter two. The differences in nitrogen content of the various parts of 
the plant have already been brought out somewhat, but they will be 
dealt with more fully in the results presented under the experimental 
portion of this bulletin. The presence of the bacteria would in itself 
be sufficient to account for these differences. 

In brief, the chemical data which have been considered, altho 
small in amount, show the relative richness in nitrogen of the nodule 
as compared with other parts of the plant. They point to the accumu- 
lation of nitrogen in the seeds, at the expense of the other parts, as 
the plant matures. That the nitrogen exists in the form of protein, 
asparagine, and other soluble forms, is also clear. The presence of 
various aliphatic and earbocyclic amino acids has been mentioned. 



^Stocklasa: Landw. Jahrb. (1895), 24, 827-863. 

-Analyzed by Aumer, 111. Agr. Exp. Sta. (unpublished data). 



1915] A Biochemical Study of Nitrogen in Certain Legimes 503 

EXPERIMENTAL 
Plan of Investigations 

The experimental studies herein reported arc fur convenience di- 
vided into two parts. Part I consists of studies made in order to de- 
termine thru which oi-gans legumes obtain their nitrogen from the air. 
Part II is concerned with an attempt to determine more definitely the 
mechanism of the reactions occurring in the fixation and assimilation of 
atmospheric nitrogen by B. 7'adicicola and legumes, a process concern- 
ing which science is greatly in the dark. This phase of the problem has 
attracted the attention of plant physiologists, physiological chemists, 
and other scientists outside the field of agricultural research. No les- 
ser chemist than Emil Abderhalden^ has written concerning it as fol- 
lows: "It would be very interesting to Ivuow the compounds into 
which these organisms convert the nitrogen. At i^resent we have no 
knowledge of this. We assume that the final substance produced is 
protein, which is then in part assimilated by the plants with the help 
of fermentation." Any light which may be throwji on this question 
will be of great value toward its final solution. 

PART I 

STUDIES TO DETERMINE THRU WHICH ORGAN LEGUMES OBTAIN 
ATMOSPHERIC NITROGEN 

For a long time it was believed that the nitrogen fixed by legume 
bacteria and assimilated by the plant was obtained thru the leaves, and 
even now many hold to this belief. Frank and Otto- in 1890 obtained 
analytical results which seemed to them to be proof of this theory. 
They believed that the bacteria were only incidentally connected with 
the process, acting perhaps as stimuli. 

The first experiment resulting in data of a contradictory nature 
was made by KossoAvitsch^ in 1891, but the results of this investigation 
were not generally accepted. Nobbe and Hiltner^ in 1899 added fur- 
ther evidence to the existing knowledge, but their conclusions, drawn 
from physiological differences, have not been substantiated by chemi- 
cal data, which seem more reliable than those of a physiological nature. 

^Abdeihalden : f*hysiological Chemistry, Trans, by Hall, 198. 
-Frank and Otto: Ber. deut. Bot. Gesell. (1890), 8, 331. 
•'Kossowitsf'h: Bot. Ztg. (1S92), 50, 697-702, 71.'5-72:?, 729-73S, 745-755, 
771-774. 

'Nobbe atul Hiltiu'r: Landw. Vers. 8tat. (1S99), 52, 455-4(i5. 



504 



Bulletin No. 179 



[March, 




O 



a 
02 



1 



Ph 



1915] A Biochemical Study op Nitrogen in Certain Legumes 505 

Experiments on this question were conducted Ijy the author in 
1911-1912. The general plan Avas the same thruout each experiment; 
the various modifications are considered under the individual experi- 
ments. 

General Plan of Experiments 

The plants used were the soybean and the cowpea. Uniform seeds 
were carefully selected and inoculated with an infusion placed directly 
in contact with them. They were then planted in beakers containing 
nitrogen-free white sand. Mineral plant food was added in solution. 
When the seedlings had developed two leaves and possessed small nod- 
ules, they w^ere carefully washed from the sand and transferred to 
the apparatus. 

The apparatus^ was arranged as follows : Woulf e bottles, placed 
inside battery jars painted black in order to obviate the influence of 
light, were connected with drier bottles, which in turn were connected 
with a gasometer. An outlet tube from each bottle was provided, 
the external end of which was immersed in water. In the first ex- 
periment two Woulfe bottles were used and in the others six. Sterile 
nitrogen-free sand containing calcium carbonate was placed in the 
Woulfe bottles and the young seedlings carefully transplanted, one 
to each. The plants were then sealed gas-tight by means of rubber 
tissue placed double thick about the stem. Rubber cement Avas also 
used to make all joints tight. 

Plant food, with the exception of nitrogen and calcium, was added 
in solution. This solution was sterilized, boiled, and cooled just pre- 
vious to its being used in order to prevent the addition of absorbed 
gases. The plant-food solutions and sterile, distilled, nitrogen-free 
water were added from the outer end of the outlet tube, with the gas 
flowing in order to avoid the possible admittance of air. The moisture 
content of the sand was maintained at about 12 percent. When the 
apparatus had been made tight, the gas was started and allowed to 
flow gently for eight to ten hours per day; at night it was entirely 
shut off. By this method the plant roots Avere kept constantly in the 
same atmosphere. 

The gas mixture used in the first three experiments consisted of 
96 to 98 percent oxygen and 2 to 4 percent carbon dioxid. For the 
purpose of comparison, air was passed thru part of the bottles in these 
experiments. The gas mixture Avas made in the laboratory, great care 
being exercised to eliminate nitrogen, air, and other impurities. The 
oxygen was made from potassium chlorate and manganese dioxid, and 
the carbon dioxid Avas generated from marble and hydrochloric acid. 
The air, Avhen used as a source of nitrogen, Avas passed thru sulfuric 
acid before entering the gasometer and after leaAang it. In order to 

'Plate V shows the apparatus in use. 



506 



Bulletin No. 179 



[ March, 




1915] 



A Biochemical Study op Nitrogen in Certain Legumes 



50' 



dispel any possible doubt as to the oxygen mixture being too strong, 
a fourth experiment was conducted in which the effect of a mixture 
made of 90 percent oxygen, 7 percent nitrogen, and 3 percent carbon 
dioxid was compared with that of a mixture made of 97 percent 
oxygen and 3 percent carbon dioxid. 

Experiment I 

In Experiment I, soybeans were used. Three plants twenty-one 
days old were placed in position on September 1, 1911, one in each of 
two Woulfe bottles and a check plant left uninclosed. Thruout the 
experiment these plants were kept out of doors during the day. The 
experiment was continued for twenty-eight days. At the end of that 
time the plants were analyzed for total nitrogen by the official Gun- 
ningi method. The average of individual analyses of twenty soybean 
seeds was taken as the criterion from which to calculate the amount 
of nitrogen fixed by the plants. The results are presented in Table 9. 

Table 9. — Fixation^ op Nitrogen by Soybeans: Experiment I 
(Eesults expressed in milligrams) 



Plant 
No. 


Treatment 


Nitrogen in 

plant at end, 

28 days 


Nitrogen 

in check 

seeds 


Nitrogen 
fixed 


1 
2 
3 


GOj + 
CO2 + O 

Air 


10.43 
10.65 
17.61 


11.4 
11.4 
11.4 


(-.97) 

(-.75) 

7.07 



^The word fixation is used in this publication in its broader sense and should 
be understood as meaning the fixation of atmospheric nitrogen by bacteria and 
the assimilation of the nitrogenous compounds formed by the plant. 

The error in Plants 1 and 2 is partially accounted for by a slight 
injury to these plants by grasshoppers and red ants. There is, how- 
ever, a small experimental error which is difficult to eliminate, as will 
be observed in the other experiments. 



Experiment II 

The experience gained in Experiment I led to the selection of 
cowpeas for the later investigations, since they are less subject to 
injury by red ants than are soybeans. Experiment II was started on 
November 23, 11)11, and continued until December 29, thirty-seven 
days. Six two-liter Woulfe bottles were planted with seedlings 
twenty-four days old. Air was passed thru three of the bottles and the 
gas mixture thru the other three. The average of individual analyses of 



'In preliminary tests the Gunning and Kjeldahl methods modified to include 
nitrates gave no higher results than the official Gunning or Kjeldahl methods. 



508 



Bulletin No. 179 



[March, 




Plate VI. — Experiment II: Plants Above Grown in Air; Those Below 

Grown in COa -f 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



509 



fifteen eowpea seedlings seventeen days old was used as a basis from 
which to calculate the nitrogen fixed by the plants during the experi- 
ment. Seedlings of this age were taken for analysis in order that the 
results of this experiment might be comparable with those of the 
others, altho the seedlings of the experiment when transplanted were 
somewhat older. 

Table 10. — Fixation of Nitrogen by Cowpeas: Experiment II 
(Results expressed in milligrams) 



Plant 
No. 


Treatment 


Nitrogen in 

plant at end, 

37 days 


Nitrogen in 

check seedlings 

at beginning 


Nitrogen 
fixed 


1 
2 
3 
4 
5 


CO2 + O 
C0„ + 
COo + O 
Air 
Air 


9.21 
13.03 

9.43 
24.84 
23.61 


7.90 
7.90 
7.90 
7.90 
7.90 


1.31 

5.13 

1.53 

16.94 

15.71 



Note. — Plant 6 was lost in distilling thru the breaking of the flask, caused 
by sand adhering to the roots. 

The fixation shown by Plant 2 is attributed to a leak discovered 
around the stem of this plant some few weeks after it had been put in 
place ; trouble was had thruout the experiment in keeping it gas-tight. 
The evident fixation in the case of Plants 1 and 3 is within experi- 
mental error; yet since these plants were twenty-four days old when 
placed in the apparatus, while the check seedlings analyzed were only 
seventeen days old, it is reasonable to assume, from the results obtained 
in the next experiment, that a part at least of the assimilation of this 
nitrogen had taken place before the seedlings were transferred. 

On the plants receiving air the nodules became well developed. 
The accompanying photograph (Plate VI), taken at the termination 
of the experiment, shows the comparative development of the roots 
and the tops grown in the gas mixture and those grown in the air. 
The most interesting part of this experiment was the very evident 
translocation exhibited by the plants growing in the mixture of carbon 
dioxid and oxygen, as shown by their color. The same phenomenon 
was observed in the later experiments and is discussed under the 
general consideration of the gas experiments. 



Experiment III 

Experiment III was conducted with cowpeas in a manner similar 
to that of the preceding experiments. It was started on March 5, 1912, 
with six seedlings seventeen days old and discontinued after eighty- 
three days, May 27, 1912. Air was passed thru three of the bottles 
and the gas mixture thru the other three. In order to obtain the best 
possible check on the results, fifteen additional seedlings of the same 



510 



Bulletin No. 179 



[March, 






Plate VII. — Experiment III: Lower Figure Showing Experiment at Begin- 
ning; Upper Figure Showing Experiment 10 Days Later 



1915] A Biochemical Study of Nitrogen in Certain Legumes 511 







.^ 










Plate VIII.— Experiment III: Upper Figure Showing Experiment 52 Days 
FROM Beginning; Lower Figure Showing Experiment at Harvest 

(83 Days) 



512 



Bulletin No. 179 



[March, 



lot as those transplanted to the Woulfe bottles, grown from seeds 185 
milligrams in weight, were analyzed individually at the beginning of 
the experiment. The results showed the presence of an average of 7.90 
milligrams of nitrogen, while the average nitrogen content of twenty 
seeds of the same weight analyzed individually equaled 6.94 milli- 
gi'ams, making an average fixation of .96 milligram of nitrogen by 
these seedlings in the first seventeen days. 



Table 11. — Fixation op Nitrogen by Cowpeas: Experiment III 
(Eesults expressed in milligrams) 



Plant 
No. 


Treatment 


Nitrogen in 

plant at end, 

83 days 


Nitrogen in 

check seedlings 

at beginning 


Nitrogen 
fixed 


1 


CO, 4-0 


9.48 


7.90 


1..58 




C0, + 


7.49 


7.90 


(-.41) 


3 


CO, + 


8.49 


7.90 


.59 


4 


Air 


Eoots 74.27 
Tops 112..59 










18(3.8(5 


7.90 


177.96 


5 


Air 


Roots 71.99 
Tops 166.03 










238.02 


7.90 


230.12 


6 


Air 


Eoots 66.71 
Tops 120.51 










187.22 


7.90 


179.32 



The figures in Table 11 show to what extent fixation took place. 
Plants 1 and 3 may have contained more than 7.90 milligrams of nitro- 
gen as seedlings, altho it cannot be proved that they did, owing to the 
impossibility of analyzing and growing the same seedling. There was . 
always another possible source of error in the dissolved nitrogen gas 
in the water used for pressure in the gasometers. 

It is well to observe that in all these experimoits the gases were 
passed thru sulfuric acid, which eliminated the possibility of ammonia 
playing any part in the fixation. This is claimed by many to occur ; 
yet the first experiment ever made for the purpose of showing that 
legumes obtain nitrogen from the air was so conducted that combined 
nitrogen was eliminated. 

The plants in the carbon dioxid and oxygen mixture were from 3 
to 4 inches in height and possessed two leaves at the end of the experi- 
ment, while those growing in the air measured from 8 to 9 inches in 
height and possessed nine leaves. 



1915] A Biochemical Study of Nitkogen in Certain Legumes 513 




Plate IX.— Experiment III: On the Left, Roots from Plant Grown in 
CO2-I-O; On the Right, Roots from Plant Grown in Axe 



514 



Bulletin No. 179 



[March, 



In order to test the viability of B. rndicicola after it had grown 
on the plant under extreme oxygen conditions, organisms were re- 
moved from the nodules of Plants 1, 2, and 3, and an infusion made in 
sterile water. Portions of this infusion were applied to cowpca seeds 
that had been sterilized and planted in sterile sand. Sterile conditions 
were maintained thruout this test. Profuse nodule formation resulted, 
demonstrating that no harmful results had been produced upon the 
organism by its long exposure to an atmosphere with a high content of 
oxygen. 

Experiment IV 

Having made certain in Experiment III that no detrimental ef- 
fects had been produced upon B. radicicola by long exposure to an 
atmosphere high in oxygen, Experiment IV was instituted in order to 
determine if there could have been any possibility of injury to the 
plants in the previous experiments from the use of gaseous mixtures 
high in oxygen. 

The plan involved a comparison of the effect of a mixture of 97 
percent oxygen and 3 percent carbon dioxid, and that of a mixture of 
90 percent oxygen, 7 percent nitrogen, and 3 percent carbon dioxid. 
The nitrogen used was obtained from the air; otherwise this experi- 
ment was similar to Experiment III. Each gas mixture was passed 
thru three of the Woulfe bottles. The experiment was begun on Sep- 

Table 12. — Fixation of Nitrogen by Cowpeas: Experiment IV 
(Kesults expressed in milligrams) 



Plant 
No. 



Treatment 



Nitrogen in 
plant 



Nitrogen in 

check seedlings 

at beginning 



First Harvest (26 Days) 



Nitrogen 
fixed 



C0, + 

N 4- CO, + O 



10.00 
14.94 



7.90 
7.90 



2.10 
7.04 



Second Harvest (28 Days) 



00^ + 

N + COo + O 



8.06 
33.51 



7.90 
7.90 



.16 
25.61 





Third Harvest (95 Days) 






1 


CO,-f 


13.97 




7.90 


6.07 


5 


N + CO, + 


Leaves 129.26 
Stems 45.17 

Tops 174.43 
Eoots 32.93 
Nodules 112.02 




7.90 






319.38 


311.48 



1915] A Biochemical Study op Nitrogen in Certain Legumes 51- 



\ - 3 





Plate X. — Experiment IV: Upper Figure Showing Experiment at Beginning; 
Lower Figure Showing Experiment 16 Days Later 



516 



Bulletin No. 179 



[March, 




Plate XL — Lxperiment IV: Lower Pigxire Showing Plants 41 Days from 
Beginning; Upper Figure Showing Plants 59 Days from Beginning 



1915] A Biochemical Study of Nitrogen in Certain Legumes 517 




Plate XII. — Experiment IV: At the Time of Harvest (95 Days) 



518 



Bulletin No. 179 



[ March, 




Plate XIII. — Experiment IV: On the Left, Roots pro.m Plant Grown in 
CO2-I-O; On the Right, Roots from Plant Grown in N-j-COj-f-O 



3 




^V'l 


1 




4 


3 




31/2 


2 




3 


3 




2% 


I 




3 



79J5] A Biochemical Study of Nitrogen in Certain Legumes 519 

tomber 7, 1912, with six cowpea seedlings eleven days old, and con- 
tinued for ninety-five days. The plants were harvested two at each 
of three periods. 

The results given in Table 12 need no explanation, tho it might 
be well to call attention to the individual differences in the plants in 
the amounts of nitrogen fixed. During the ninety-five days of the ex- 
periment, Plant 5 fixed fifty-one times as much nitrogen as Plant 1. 
The following comparison between the growth of these two plants is 
of interest. 

Plant 1 attained a total height of 5 inches, possessed one leaf, and 
on the roots were counted 60 nodules. Plant 5 reached a height of 61 
inches ; its leaves measured as follows : 

2 measured 5 inches along midrib, 4 inches at base 
" " 3-31/^ " " " 

. ; ) J ^ 1 ■> 1 1 ■> 1 

11 11 O]/ > > ) ) 11 

I > 11 2 " " ' ' ' ' 

II 11 -11/ 11 11 J 1 

11 ! ) 2 111111 

Several pods were formed, as may be seen by reference to Plate 
XII, one of which measured 4I/2 inches in length and was partially 
filled with seeds. The roots were so large that the Woulfe bpttle had 
to be broken in order to obtain them. The plant possessed 32 large 
nodules, 46 medium to large, 66 medium, and 144 small ; 288 in all. 

Experiment by Kossowitsch 

Reference has been made to a laboratory experiment conducted 
by Kossowitsch in the summer of 1891 (see page 503). As his work 
has been accei)ted by some and ignored by others, it is of particular 
interest. 

Peas were started in a mixture of four-fifths sand and one-fifth 
soil in which peas had been grown the year before. When the plants 
had developed good nodules, they were transferred to jars containing 
nitrogen-free sand. In some cases the roots were enclosed and in others 
the tops, a similar means being used in each case to make the joints 
air-tight. Over the tops of the jars bell-jars were placed. These 
were connected thru drier bottles with a gasometer. Because of mois- 
ture collecting in the bell- jars, absorbents were used to keep the 
atmosphere normal. The gas mixtures used were hydrogen and 
oxygen in some cases ; hydrogen, oxygen, and carbon dioxid in some ; 
and air in others. Combined nitrogen was also used to check up 
the possible abnormal condition due to the necessary manner of 
experimentation. Great accuracy was displayed in arranging the ap- 
paratus and in analyzing the gases. 



520 



Bulletin No. 179 



[ March, 



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1915] A Biochemical Study op Nitrogen in Certain Legumes 521 

A study of the results of Kossowitsch 's experiment, given in Table 
13, discloses very little data bearing strictly on the question, thru 
what organs plants obtain atmospheric nitrogen. Plants 1 and 2 
tend to show that nitrogen cannot be obtained thru the leaves. Plant 
2 shows a fixation, but it may be within experimental error. Plants 3, 
4, 6, and 10 may be eliminated, as assimilation should have taken place 
in these cases. Plant 5, the leaves of which were in hydrogen, oxygen, 
and carbon dioxid, fixed nitrogen, but Kossowitsch states that this re- 
sult is not reliable, owing to the leaves not having been sufficiently iso- 
lated. Plants 7, 8, and 11 show normal results, and, like Plant 9, 
indicate, aside from 'the experimental error, that the nitrogen must be 
brought into contact with the roots in order to effect fixation. Plant 
12, the leaves of which were enclosed in hydrogen, oxygen, and carbon 
dioxid, also showed a fixation. Thus it will be seen that the data 
bearing on the question were obtained from only seven plants. 

Somewhat related to this problem is the work of Nobbe and Hilt- 
ner,i who grew lupines in solutions. They thought that it was neces- 
sary to raise the nodules above the solution in order to obtain a 
fixation, and this seemed to indicate to them that legume plants obtain 
atmospheric nitrogen thru their roots rather than thru their tops. 

General Consideration op Gas Experiments 

The plants in the mixture of carbon dioxid and oxygen usually 
developed two and sometimes three leaves before they seemed to be 
checked in their growth. Soon an interesting translocation set in. 
Each plant removed the nitrogen from the lower leaves and developed 
a new leaf of a normal green color. The green of the old leaves disap- 
peared from the margins first; soon the whole leaves became yellow, 
and shortly dropped from the plant. This process repeated itself un- 
til there was not nitrogen enough left in a translocatable form to give 
green color to another leaf, when a pale green or even a yelloAV leaf 
appeared. The duration of this process was remarkably long in some 
of the plants. The appearance of the plants was in every case of 
especial note, even to a layman. 

It should be emphasized that no combined nitrogen was present 
or could have been assimilated in these experiments. The slight amount 
of nitrogen reported as fixed by the plants in carbon dioxid may have 
been either actual fixation or experimental error. From close observa- 
tion and study made in order to reduce this error, it would seem that 
its only possible source, disregarding Plant 2 in Experiment II, 
already mentioned, would be the dissolved air in the water used for 
pressure in the gasometers. This was unavoidable. It may also account 
for the error in Kossowitsch 's experiment. Plant 1 in Experiment IV 

*See page 503, note 4. 



522 Bulletin No. 179 [March, 

would seem to indicate that the above view is correct, as it showed a 
content of 6 milligrams of nitrogen after having remained under ex- 
periment for ninety-five days. In this length of time there were a 
great many changes of the gasometers and the use of a large amount 
of water, which would tend to increase the atmospheric nitrogen and 
thus perhaps contaminate the other gas mixtures. The nodules on all 
the plants were normal. On Plant 5, Experiment IV, they were very 
large, and the spongy sutures were highly developed as if to present 
as large an absorbing surface as possible. 

Finally, it should be noted that had the plants growing in the 
mixture of carbon dioxid and oxygen possessed any ability to take in 
nitrogen thru their leaves, they should have made as good a develop- 
ment as those growing in the air, and those in the oxygen, nitrogen, 
and carbon dioxid mixture. 

Practical Application of Results 

The practical application of the results obtained in these experi- 
ments would appear to rest in the proper aeration of the soil in order 
that greater amounts of nitrogen may be fixed. As the plants obtain 
their nitrogen thru their roots, it is essential that the soil contain 
plenty of air at all times. 

PART II 

RELATIVE PERCENTAGES OF NITROGENOUS COMPOUNDS IN THE 

VARIOUS PARTS OF THE SOYBEAN AND COWPEA AT 

DEFINITE PERIODS OF GROWTH 

It has seemed advisable to determine, if possible, in what forms 
atmospheric nitrogen exists in the various parts of the legume, for it 
is believed that this knowledge would throw much light upon the whole 
process of fixation. To do this, various compounds in the nodules, 
roots, and tops were determined at definite periods in the growth of 
the soybean (Glycine Jiispida, Maxim) and the cowpea {Vigna un- 
guiculata, Walp). As it would be quite impossible to determine all 
these compounds separately, they were grouped and determined as 
follows, — total, insoluble, and soluble nitrogen. In the soluble-nitro- 
gen group was included the nitrogen precipitated by P.T.A. and 
Other nitrogen. The constructive and destructive metabolisms in the 
plant doubtless give rise to other nitrogen compounds than pure pro- 
tein; so it is quite possible that in addition, proteoses, peptones, pep- 
tides, acid amides, amino acids (mono and di), guandine residues, pig- 
ment nitrogen, alkaloids, ammonia, nitrites, nitrates, and other nitro- 
gen compounds may have been present. 

Because of the great importance of controlling conditions under 
which experimental plants are grown, all the factors in this work were- 
controlled except the influence of sunlight. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



523 



Methods Employed in the Growth and Preparation of Samples 

Plants Used. — The soybean and the cowpea were selected for this 
work because of their adaptability to the conditions under which it 
was necessary to carry on the investigations. The rapidity and habit 
of growth, as well as the nodule formation of these plants, makes them 
especially desirable for experiments of this sort. The cowpea is an im- 
portant crop, especially in southern Illinois, while the soybean is being 
grown more and more each year in central and nortliern Illinois. The 
seed used in all the experiments conducted was produced in 1910 on 
some of the experimental fields of this station. The soybeans were of 
the Medium Green variety, the cowpeas, the Whippoorwill. 

Metliods Used in Growing the Plants. — 4.7 kilos of pure nitrogen- 
free sand, in which 10 grams of chemically pure calcium carbonate had 
been thoroly mixed, were placed in each of a number of 6-inch cylin- 




Plate XIV. — Typical Jar of Five Cowpeas Being Grown for Samples 



524 Bulletin No. 179 [March, 

drical glass battery jars. These jars were painted black in order to 
keep the light from the roots and check the growth of algae. A mois- 
ture content of 12 percent was maintained thruout the growth of the 
plants. This was done by weighing each jar at least every week, some- 
times every four days, and adding sufficient nitrogen-free, distilled 
water 1 to restore the original weight of the jar. During the interim 
each jar was given the same amount of water. 

Mineral plant food in solution was applied once a week. Each 
jar was given the following solutions: 

10 cc. each of — 

25 grams CaH^ (P04)2 per 2500 cc. water 

20 grams MgS04 per 2500 cc. water 

50 grams K2SO4 per 2500 cc. water 

1 cc. of — 

.1 gram FeClj per 250 cc. water 

These amounts were diluted with water and added at the same 
time that the plants were made up to weight. The jars in several 
series were kept out of doors during the pleasant days, but all plants 
were kept in the greenhouse during the night. 

Planting and Inoculation. — Five seeds of average size were se- 
lected and planted in each jar. In the earlier work, in order to insure 
a proper germination of the five seeds and avoid the possibility of some 
decaying and leaving organic matter in the sand, moistened filter 
papers were placed over the seeds until germination was assured, when 
the seeds were covered with one-half inch of sand. Later this method 
was found unnecessary owing to the excellent germination of the seeds ; 
and by starting more jars than needed in the series, the required num- 
ber containing five plants was insured. By always covering the seeds 
with the same amount of sand, more uniform plants were secured. 

Inoculation was attained by the following method: Plants were 
grown in a soil in which they would form nodules. These nodules 
were then removed from the plant, thoroly washed, and dipped in 
alcohol. After burning off the alcohol by passing the nodules thru a 
flame, they were crushed in sterile distilled water. The inoculum was 
then diluted to about a liter and 5 cc. applied to each seed just be- 
fore it was covered with sand. When this method of infection was 
carried out, no failures were experienced and an abundance of nodules 
was always obtained. 

Uninoculated material was obtained by planting a few jars sim- 
ilarly to the inoculated, except that the seeds were dipped in 
alcohol and the alcohol then burned off, and that a sterile spatula was 
used in planting. The sand in these jars was not sterilized, but the 
jars were placed in vessels of water in order to prevent possible infec- 



^Whenever water is mentioned in this publication nitrogen-free distilled 
water is always meant, unless otherwise stated. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 525 

tion by ants and red spiders. This method gave very good results; 
in no case did inoculation occur. 

Harvesting Samples. — As soon as the seed coats were free from 
the young seedlings, they were removed from the jars, labeled accord- 
ing to the jars from which they came, and later were analyzed with 
the roots from that jar. In like manner the cotyledons and all the 
leaves which had dropped were analyzed with the tops from the cor- 
responding jar. 

The periods at which the plants were harvested were regulated ac- 
cording to the development of the leaves. Harvests were made from 
jars as nearly uniform as possible, in most cases selected in triplicate. 
The jars were taken to the laboratory, where the tops of the plants 
were cut one inch above the surface of the sand and placed in a suit- 
able receptacle to air-dry. A stream of water was then carefully di- 
rected on the jars in order to wash the sand from the roots. After all 
the visible nodules had been carefully removed with forceps, counted, 
and placed away to air-dry, the roots were also placed away to air-dry. 

Laboratory numbers were given as follows : odd hundreds to soy- 
bean series, even hundreds to cowpeas ; the tens to the number of the 
harvest; and the units 1, 2, 3, to tops; 4, 5, 6, to roots; and 7, 8, 9, to 
nodules. Thus, 129 refers to soybean nodules of the second harvest. 
The roots of the same are numbered 126, and the tops, 123. 

Preparation of the Sample. — After complete air-drying, all the 
samples were ground so that they would pass thru a 100-mesh sieve. 
No difficultities were experienced in the grinding except with that 
part of the stem which extends a little above and below the surface of 
the sand. At this place where the plant needs the greatest strength to 
sustain its upright position, the fiber is very tough. The difficulty in 
grinding this part of the stem was overcome by adding some grains of 
pure sand before grinding the tops and roots. All possible care was 
exercised in grinding, yet some slight loss was inevitable. This was 
especially true with the soybean leaves, as they are pubescent and small 
hairs will sometimes float in the air. However, it is doubtful whether 
the error due to this loss was relatively as great as that due to chemi- 
cal manipulation. As the results given in the tables all refer to milli- 
grams of nitrogen per jar, the presence of sand in some samples does 
not affect them. After the samples had been ground they were placed 
in air-tight jars until needed for analysis, when the total weight of 
each was taken. 

Analytical Methods 

Total Nitrogen. — When determining total nitrogen, duplicate 
subsamples for each determination on each sample were weighed out 
at the same time in order to avoid error from possible moisture 
changes. For the tops and roots .5 gram was taken and for the 



526 Bulletin No. 179 [March, 

nodules .1 or .2 gram. The total nitrogen was determined by the Jodl- 
bauer method.^ Glass beads were used to prevent bumping in digest- 
ing. The hydrochloric acid and the ammonium hydroxid used in 
titrating were one-tenth normal. Lacmoid was used thruout as the 
indicator. The roots were not so easily oxidized as the other parts, 
but the use of permanganate to complete the oxidization was avoided, 
except in the case of a very few determinations. 

Insoluble Nitrogen. — In determining insoluble nitrogen, a .2-gram 
sample was weighed out and placed in a 400-cc. shaker bottle, 150 cc. 
of water was added, and the bottle was then placed in a mechanical 
shaker for three hours. In some cases a smaller sample was used, es- 
pecially with the nodules, and sometimes with the roots ; in these cases 
a smaller amount of water was used. The contents of the bottle were 
then filtered onto an S. & S. filter paper and platinum cone, suction 
being used when necessary. The residue and filter paper were trans- 
ferred to a Kjeldahl flask and the insoluble nitrogen determined by 
the Kjeldahl method. 

Soluble Nitrogen. — The total soluble nitrogen was obtained by ad- 
ding P.T.A.2 and Other nitrogen. In the few cases where distillation 
was made with sodium hydroxid, the nitrogen obtained is also in- 
cluded under the soluble nitrogen. 

Nitrogen by Distillation with NaOH. — In several of the harvests 
of the first two series, the filtrate from the insoluble nitrogen was dis- 
tilled with sodium hydroxid. The nitrogen obtained by this treatment 
is reported as nitrogen by sodium hydroxid. This nitrogen may rep- 
resent various compounds, as will be explained later. 

P.T.A. Nitrogen. — ^In order to determine P.T.A. nitrogen, the 
filtrate from the insoluble residue was made up to 200 cc. with water. 
Five grams of concentrated sulfuric acid per 100 cc. of solution were 
added and then 10 cc. of a solution containing 20 grams of P.T.A. 
and 5 grams of sulfuric acid per 100 cc. of water. In the beginning 
30 cc. of P.T.A. solution was used ; later this amount was reduced to 
10 cc. and in some cases to only 5 cc. with the roots. This precipita- 
tion was made in the cold and the solution allowed to stand for forty- 
eight hours in order to obtain a complete precipitation of arginine. 
At the end of that time the precipitates were filtered off thru S. & S. 
filter papers and washed with a solution containing 2.5 grams of 
P.T.A. and 5 grams of sulfuric acid per 100 cc. Later, washing was 
carried out by using part of the mother liquor. The precipitates on 
the filters when thoroly washed and dried were transferred to Kjel- 
dahl flasks, and the nitrogen determined by the Kjeldahl method. 



^The Jodlbauer method was used at first, as it was thought nitrates might 
be present, but later it was discontinued except for determining total nitrogen. 
^See note to Table 4, page 500. 



1915] A Biochemical Study of Nitrogen in Certain Legumes 527 

Other Nitrogen. — In determining Other nitrogen, the filtrate 
from the P.T.A. precipitate, after having I een evaporated to about 
30 ec. and then made up to 50 cc., was divided into two parts in the 
first series, and a Kjeldahl determination made on one half and an 
amino-nitrogen determination on the other. This proved unsatisfac- 
tory because of the small amount of nitrogen present in the filtrate for 
an amino-nitrogen determination. Owing to the excess of P.T.A. in 
this filtrate, serious bumping occurred during digestion. Altho glass 
funnels were placed in the necks of the flasks to prevent loss, some de- 
terminations were lost. Digestion was continued for four to seven 
hours with this filtrate. 

A few modifications of the above methods are considered in con- 
nection with the series in which they occurred. In all the analytical 
work the reagents were carefully and constantly checked up for nitro- 
gen, altho the methods were applied under the same conditions at all 
times. 

Discussion of Some of the Methods ITsed 

The insoluble nitrogen represents certain proteins and probably 
other insoluble nitrogenous compounds. The nitrogen obtained by dis- 
tillation of the filtrate from the above might represent nitrogen from 
acid amides or volatile organic bases or basic amino nitrogen (arginine 
and cystine). The presence of volatile organic bases was eliminated 
by qualitative tests. Thus it would seem that the nitrogen found by 
the distillation represents an amide or basic nitrogen. The presence 
of asparagine in soybean seedlings has already been pointed out, but 
it cannot be assumed that the nitrogen thus determined was aspara- 
gine, as arginine and cystine may have been present. 

P.T.A., as already stated, precipitates various nitrogenous com- 
pounds. The reagent, however, does not completely precipitate alka- 
loids, peptones, proteoses, peptides, and diamino acids (basic nitro- 
gen) ; further, Van Slyke^ has shown that the diamino acids are ap- 
preciably soluble in this reagent. Osborne and Harris^ have demon- 
strated that P.T.A., while subject to various criticisms, nevertheless, 
when used under constant conditions, gives very good comparative 
results. 

The filtrate from the P.T.A. precipitate contains all soluble nitro- 
gen not precipitated by P.T.A, This nitrogen may be made up of 
amino acids, pigment nitrogenous compounds, and possibly other ni- 
trogenous compounds. 

Qualitative Tests 

A large number of plants and parts thereof, grown under the 
same conditions as those harvested for quantitative determinations 



^Van Slyke: Jour, Biol. Chem, (1911-12), 10, 15-56. 

''Osborne and Harris: .Tour. Am. Chem. Soc. (1903), 25, 32.'^-.1.'?.'?. 



528 



Bulletin No. 179 



[March, 



were tested for ammonia, nitrites, and nitrates. Nessler's reagent was 
used to test for ammonia and diphenylamine sulfuric acid for nitrites 
and nitrates. All these tests were negative. Ten grams of tops were 
treated with 800 cc. of water in a liter flask. The solution was boiled 
but no ammonia was obtained. Upon the treatment of the filtrate 
from this solution with sodium hydroxid, a large amount of ammonia 
was found but no volatile organic bases, as the distillate gave the typi- 
cal test for ammonium chlorid when absorbed in hydrochloric acid 
and left no carbonaceous residue. Zinc sulfate gave no precipitate in 
this filtrate. The filtrates from the P.T.A. precipitates were tested in 
the Van Slyke apparatus with results which indicate that some of the 
nitrogen in the filtrate was in the form of primary amines. 

Since the completion of these experiments, a study of the total 
amino nitrogen in the seedlings of the Alaska pea, as obtained by the 
Van Slyke apparatus, has been reported by Thompson. ^ His results 
indicate that as high as 43.3 percent of the total nitrogen may be in 
the form of primary amines. The percentage of amino nitrogen found 
in the seed was only .088, increasing in the seven-day seedlings to 
28.27. 

Series 100 (Soybeans) 

The conditions under which the soybeans used in Series 100 were 
grown have already been explained (see page 523). Their develop- 
ment at each of the five harvests is shown in Table 14. 

Table 14. — Development of Soybeans: Series 100 



Planted 


Harvested 


Age, 
days 


Leaves and pods 
per plant 


Height, 
inches 


Nodules 
per 15 
plants 


Mar 


18, 


1911 


Apr. 


25 


38 


4 leaves 


6 


265 


} } 


} J 


>> 


May 


10 


53 


6 " 


11 


1 061 


y > 


} ) 


> y 


May 


17 


60 


8 " 


14 


987 


} } 


) y 


y y 


May 


24 


67 


10-12 leaves; an 
average of 5.4 pods 


16-17 


1 154 


>> 


y y 


y y 


May 


31 


74 


10-12 leaves; beans 
formed in pods 


16-17 


1 354 



The total nitrogen determinations in the various parts of the 
plants of this series at different stages of growth, together with the 
nitrogen fixed at each of these stages, are given in Table 15. The 
amount of the nitrogen fixed was determined by subtracting, from the 



^Thompson: Jour. Am. Chem. See. (1915), 37, 230-235. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



529 



total nitrogen found, the average nitrogen content of five soybean 
seeds as shown by the individual analyses of twenty seeds. Nearly all 
the figures in this table represent the average of six determinations. 

Table 15. — Total Nitrogen in Various Parts of Soybeans and Fixation 
AT Different Periods: Series 100 

(Milligrams per jar of five plants) 



Harvest 


Lab. 
Nos. 


Nitrogen 
in tops 


Nitrogen 
in roots 


Nitrogen 
in nodules 


Nitrogen 

in whole 

plants 


Nitrogen 
in seeds 

57.30* 


Nitrogen 
fixed 


1 


111-112 
114-115 
117-118 


87.10 


13.35 


28.04 


128.49 


71.19 


2 


121-129 


204.59 


22.70 


47.10 


274.39 


57.30 


217.09 


3 


131-139 


286.91 


48.44 


82.95 


413.30 


57.30 


356.00 


4 


141-149 


356.52 


40.15 


60.40 


457.07 


57.30 


399.77 


5 


151-159 


247.82 


30.82 


54.56 


333.20 


57.30 


275.90 



*It would be preferable to use the analyses of uninoculated plants as a check 
rather than the analyses of seeds. 



These figures need very little explanation. The results of the first 
four harvests show a gradual increase in the amount of nitrogen fixed. 
The low results obtained at the last harvest are in accord with the 
results of Wilfarth and Wimmer^ and Penny and MacDonald.^ 

The separation of the nitrogen compounds into the various groups 
was carried out in this series as follows : The whole eample was dried 
for four hours at 50° C. both before and after grinding. The subsam- 
ples were weighed out and placed in 250-cc. beakers ; 100 cc. of Avater 
was then added and the whole heated to boiling and filtered while hot. 
Suction was used in filtration, and the washing was done with 50 cc. 
of hot water. The residue was Kjeldahlizcd as usual. The filtrates 
from some of the harvests were treated with 10 cc. of sodium hydroxid 
and distilled. The residual liquid in the Kjeldahl flask was trans- 
ferred to a 350-cc. beaker and the excess alkali neutralized with sul- 
furic acid, after which the regular P.T.A. method was applied. The 
precipitates obtained by P.T.A. were characteristic in their behavior. 
After the reagent had been added some one or two hours, a volumi- 
nous grayish white precipitate appeared, sometimes colored a yellow- 
ish green, and very graduallj^ settled to the bottom in a very thin 
layer, leaving the supernatant liquid yellowish green in the case of 

^Wilfarth and Winimer: Landw. Vers. Stat. (1906), 63, 1-70, 
^'Penny and MacDonald: Del. Agr. Exp. Sta. Bui. 86, 35. 



530 



Bulletin No. 179 



[March, 



the tops, slightly straw colored to colorless in the case of the roots, 
and colorless in the case of the nodules. 

The amount of the precipitate from the determination with the 
tops was much greater than that with the roots and nodules. Caution 
was exercised in all the determinations not to allow losses or changes 
due to bacterial action. A few drops of chloroform were placed on the 
filter and in the filtrate when the determinations were sufficiently long 
to be liable to bacterial action. 

The results of the separations are shown in Table 16. Here again, 
as in the case of the total nitrogen determinations, most of the figures 
represent the average of two determinations made upon samples from 
each of three jars. The total soluble nitrogen was obtained by the 
addition of the various determined soluble forms. The total nitrogen 
reported in the last column is the sum of all the separations made. 
These results will be discussed later with those of the other series. 

Table 16. — Nitrogen Separations: Series 100 (Soybeans) 
(Milligrams per jar of five plants) 



Har- 
vest 


Lab. 

Nos. 


Part 


Insol- 
uble 
nitro- 
gen 


Total 
solu- 
ble 
nitro- 
gen 


NaOH 

nitro- 
gen 


P.T.A. 
nitro- 
gen 


Other 
nitro- 
gen 


Total 
nitro- 
gen 


1 


111-112 
114-115 
117-118 


Tops 
Eoots 
Nodules 


61.52 

8.90 

15.72 


24.39 

5.00 

11.61 


.... 


4.16 

.85 

3.54 


20.23 
4.15 
8.07 


85.91 
13.90 
27.33 


2 


121-123 
124-126 
127-129 


Tops 
Eoots 
Nodules 


135.15 
15.49 
32.83 


37.99 

5.67 

16.03 




8.11 

.48 

9.66 


29.88 
5.19 
6.37 


173.14 
21.16 

48.86 


3 


131-133 
134-136 
137-139 


Tops 
Roots 
Nodules 


146.79 
27.03 
47.95 


140.12 
16.42 
35.00 


.... 


25.63 

.93 

18.55 


114.49 
15.49 
16.45' 


286.91* 
43.45 
82.95* 


4 


141-143 
144-146 
147-149 


Tops 
Roots 
Nodules 


183.35 
26.14 
31.77 


134.26 
12.93 

27.27 


17.86 
2.49 
2.38 


25.96 

.85 

15.35 


90.44 
9.59 
9.54 


317.61 
39.07 
59.04 


5 


151-153 
154-156 
157-159 


Tops 

Roots 

Nodules 


151.68 
21.55 
29.23 


95.32 
14.38 
27.21 


12.02 
1.34 

2.00 


29.31 

1.38 

12.13 


53.99^ 
11.66 
13.08 


247.00* 
35.93 
56.44 



'Taken from Table 15. 
'Obtained by difference. 



The accompanying graph (Plate XV) shows clearly the relation- 
ship in which the soluble and the insoluble nitrogen exist at the various 
periods of growth. In this series the first harvest was not made for 
thirty-eight days, and therefore the period at which fixation began is 
not shown. Attention has already been called to the low nitrogen fixa- 



1915] A Biochemical Study of Nitrogen in Certain Legumes 531 



Soluble ^/md Insoluble Nit t^ooen 
IN To¥^5 Tfoora /^nd Nodules ofSoybe/jns 

JJlFFEJ^E-NT 'PEFflOnS OF JJe VE tOrr/E NT 

Ser,es /OO 

LtOtND 

doLuBLE □ Insoluble ■ 




ToF'-b 



J Jj 



Tfoor5 



isn^, "HDa. (.0 Ua.. Li n^^ 74- Lou. 

Nodules 



Plate XV 



532 



Bulletin No. 179 



[ March, 



tion found at the last harvest in this series. More results are necessary 
to confirm the supposition of a possible loss in the total nitrogen. 

The importance of the amount of soluble nitrogenous compounds 
at the various stages of growth has not yet been emphasized. Prelimi- 
nary studies have shown this soluble nitrogen to be much more rap- 
idly converted into ammonia and nitrates than the insoluble nitrogen. 
This would seem to have a direct bearing upon practical methods of 
handling leguminous crops in rotations when the shortest time must 
intervene between the turning under of the legume and the planting 
of the next crop. It would seem desirable to choose that period when 
the greatest amount of soluble nitrogen exists. 

Series 500 (Soybeans) 

Soybeans were used in Series 500. The plants were placed out of 
doors during pleasant days in August and September. From Table 17, 
showing the development at the four harvests, it will be seen that 
these plants made a more rapid growth than those in Series 100. 

Table 17. — Plant Development: Series 500 (Soybeans) 



Planted 


Harvested 


Age, 


Leaves and pods 


Height, 


Nodules per 






days 


per plant 


inches 

7 


15 plants 


Aug. 8, 1911 


Aug. 22 


14 


3 leaves 


30^ 


J > } ) } y 


Aug. 30 


22 


4 " 


10 


278 


J J J J > J 


Sept. 8 


30 


6 " 


14 


370 


1 ) > ) ) } 


Sept. 19 


41 


7-8 " ; 6 one- 
inch pods 


14 


247 (large) 



Tor ten plants. 

The total nitrogen determinations for this series are shown in 
Table 18. These results agree with those shown in Table 15, altho 
they represent earlier stages of development. 

Table 18. — Total Nitrogen in Various Parts of Soybeans and Fixation 
at Different Periods: Series 500 

(Milligrams per jar of five plants) 



Har- 
vest 


Lab. 
Nos. 


Nitro- 
gen in 
tops 


Nitro- 
gen in 
roots 


Nitro- 
gen in 
nodules 


Nitro- 
gen in 
whole 
plants 


Nitro- 
gen in 
seeds 


Nitro 

gen 

fixed 


1 
2 
3 
4 


511-519 
521-529 
531-539 
541-549 


46.99 
47.32 
96.96 

205.40 


8.50 
11.08 

9.76 
18.65 


.35 
11.05 
17.81 
26.98 


55.84 

69.45 

124.53 

251.03 


57.30* 
57.30 
57.30 
57.30 


(-1.46) 
12.15 
67.23 

193.73 



*This figure is approximate rather than exact. See note to Table 15, page 529. 



The separations in this series differed from those in Series 100 
in that in this case a cold-water extract was made. The sub-samples 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



533 



were placed in shaker bottles and the same amount of water added 
as in the former series; the bottles were then put in a mechanical 
shaker for three hours. This method was considered to be more in ac- 
cord Avith natural conditions than the one used in the former series. 
The nodules were filtered thru a diatomaceous earth filter. 

The figures in Table 19 were obtained in the same manner as those 
in Table 16. 

Table 19.- — Nitrogen Separations: Series 500 (Soybeans) 
(Milligrams per jar of five plants) 



Har- 
vest 


Lab. 

Nos. 


Part 


Insol- 
uble 
nitro- 
gen 


Total 
soluble 

nitro- 
gen 

25.90 
4.48 


NaOH 
nitro- 
gen 


P.T.A. 

nitro- 
gen 


Other 
nitro- 
gen 


Total 
nitro- 
gen 


1 


511-512 
514-515 
517-518 


Tops 
Eoots 
Nodules 


19.48 
4.42 


3.16 
.95 


3.31 
.33 


19.43 

3.20 


45.38 
8.90 


2 


521-523 
524-526 
527-529 


Tops 
Eoots 

Nodules 


29.60 
7.97 


17.34 

2.14 


1.18 
.00 


4.96 

.47 


11.20 
L67 


46.94 
10.11 


3 


531-533 
534-536 
537-539 


Tops 

Eoots 

Nodules 


70.04 

8.56 

16.75 


31.64 
2.29 
1.29 


2.67 
.00 
.00 


6.48 
.70 
.33 


22.49 

1.59 

.96 


101.68 
10.85 
18.04 


4 


541-543 
544-546 
547-549 


Tops 
Eoots 
Nodules 


115.39 
13.16 

22.55 


76.11 
7.35 
2.46 




10.68 

2.06 

.26 


65.43 
5.29 
2.20 


191.50 
20.51 
25.01 



Series 700 (Soybeans) 

Soybean seeds Avere planted on September 6 for this scries, but 
owing to their damping off, the jars were replanted on September 13. 
The seedlings that damped off were tested for ammonia, nitrites, and 
nitrates, with negative results. The conditions of the plants at the 
various harvests are shown in Table 20. 



Table 20. — Plant Development: Series 700 (Soybeans) 



Planted 


Harvested 


Age, 
days 


Leaves 
per plant 


Height, 
inches 


Nodules per 
15 plants 


Sept. 13, 1911 


Sept. 25 


12 


2 leaves 


5 


Nodules pres- 
ent too small 
to remove 


J ) J ) > J 


Oct. 6 


23 


3 leaves par- 
tially devel- 
oped 


8-9 


254 


) 1 ) > } > 


Oct. 14 


31 


5 leaves 


10-12 


229 


if > > ) ) 


Oct. 25 


42 


7 leaves 


12 


288 



534 



Bulletin No. 179 



[March, 



The figures reported in Table 21 present the average of duplicates 
of composite samples which included the whole of the material from 
three jars. As will be seen, these results are concordant with those of 
the two series already considered. 

Table 21, — Total Nitrogen in Various Parts of Soybeans and Fixation 
AT Different Periods: Series 700 

(Milligrams per jar of five plants) 



Har- 
vest 


Lab. 

Nos. 


Nitro- 
gen in 
tops 


Nitro- 
gen in 
roots 


Nitro- 
gen in 
nodules 


Nitro- 
gen in 
whole 
plants 


Nitro- 
gen in 
seeds 


Nitro- 
gen 
fixed 


1 
2 
3 
4 


711-719 

721-729 
721-739 
741-749 


40.08 

48.00 

68.32 

110.70 


8.88 

8.93 

11.38 

20.55 


7.21 

8.97 

17.22 


48.96 
64.14 

88.67 
148.47 


57.30^ 
57.30 
57.30 
57.30 


(-8.34) 

6.84 

31.37 

91.17 



^See note to Table 15, page 529. 

The figures in Table 22 showing the nitrogen separations were ob- 
tained in the same manner as those reported for the nitrogen fixation 
in Table 21 ; that is to say, they are the averages of duplicates of com- 
posite samples. 

Table 22. — Nitrogen Separations: Series 700 (Soybeans) 
(Milligrams per jar of five plants) 



Har- 
vest 


Lab. 
Nos. 


Parts 


Insol- 
uble 
nitro- 
gen 


Solu- 
ble 

nitro- 
gen 


P.T.A. 

nitro- 
gen 


Other 
nitro- 
gen 


Total 
nitro- 
gen 


1 


711 
714 
717 


Tops 
Boots 

Nodules 


9.94 
3.87 


28.03 
4.95 


4.70 
.39 


23.33 
4.56 


37.97 

8.82 


2 


721 

724 

727 


Tops 
Boots 

Nodules 


23.64 
5.50 


21.56 
2.55 


11.96 

.77 


9.60 

1.78 


45.20 
8.05 


3 


731 
734 
737 


Tops 
Boots 

Nodules 


26.50 
7.30 
6.30 


29.05 
3.76 
1.79 


13.75 

1.10 

.10 


15.30 
2.66 
1.69 


55.55 

11.06 

8.09 


4 


741 
744 
747 


Tops 
Boots 
Nodules 


46.45 
10.67 
14.52 


61.97 
6.31 
2.70 


23.75 
.61 
.00 


38.22 
5.70 
2.70 


108.42 
16.98 
17.22 



The close agreement of the results of Series 700 and 500 is very 
evident in the data presented. By reference to the accompanying 
graph (Plate XVI) it will be seen more easily than in the tabular 
form that the soluble nitrogen predominates in the early growth of the 
seedling. The amount decreases during this period, however, while 



1915] A Biochemical Study of Nitrogen in Certain Legumes 



535 



JJiFFETfE N T 'PeT^IOVd or JJe UdLOP'^E N7 
•5ol uBl E I I li^bOL UI3L C ^1 



6e^'f ^ 7oo 




Tq-p^ 



?7bo76 



^ 3 Ho. 



3IJ]a,. '^BJla 



NODUL t 5 



5f T'ES t 



a J 




7^^ 



/Too 7" 3 




/-? Da 



?2 Ba. 3QD^ 

Nob oLt b 
Plate XVI 



4/Z7j 



536 



Bulletin No. 179 



[March, 



the insoluble nitrogen always increases. This is true of the roots as 
well as the tops. It is during the period between the twelfth and the 
twenty-second days that nitrogen fixation begins, according to meas- 
urements by the most accurate chemical methods. Detailed studies 
are now being made of the exact time when fixation begins. 

Series 600 (Cowpeas) 

Cowpeas were used for Series 600. Owing to the smaller nitro- 
gen content of cowpea seeds, the plants show a need of nitrogen much 
sooner than soybeans, and are therefore perhaps better suited to ex- 
perimentation of this sort. The plants grown in this series are com- 
parable with the soybeans in Series 500 as regards time and conditions 
of growth. The data in Table 23 show the development of the cow- 
peas when harvested. 



Table 23. — Plant Development: Series 600 (Covppeas) 



Planted 


Harvested 


Age, 
days 


Leaves 
per plant 


Height, 
inches 


Nodules per 
15 plants 


Aug. 8, 1911 

} y J ) } } 

> > ) > 7 7 
7 > 7 ; 7 J 


Aug. 22 
Aug. 30 
Sept. 8 
Sept. 19 
Oct. 5 


14 

22 
30 
41 
58 


3 leaves 

4 " 

5 " 
6-7 " 

8 " 


7 
10 
12 

13-14 
14 


450 (very small) 

892 (small) 
1074 
2062 
1992 



The results given in Table 24 show a fixation of nitrogen at the 
end of fourteen days from the time the seeds were placed in the sand. 
The increased fixation is greater with the cowpeas in this series than 
with the soybeans in the corresponding series (500). The other gen- 
eral tendencies appear to be the same as in the other series. 

Table 24.— Total Nitrogen in Various Parts of Cowpeas and Fixation 
AT Different Periods: Series 600 

(Milligrams per jar of five plants) 















Nitrogen 




Harvest 


Lab. 

Nos. 


Nitrogen 
in tops 


Nitrogen 
in roots 


Nitrogen 
in nodules 

1.96 


Nitrogen 

in whole 

plants 

40.12 


m umn- 

oculated 

plants 


Nitrogen 
fixed 


1 


611-619 


29.11 


9.05 


36.74* 


3.38 


2 


621-629 


45.46 


10.25 


9.22 


64.93 


36.74 


28.19 


3 


631-639 


91.63 


16.64 


18.52 


126.79 


36.74 


90.05 


4 


641-649 


188.40 


30.28 


42.33 


261.01 


36.74 


224.27 


5 


651-659 


439.64 


73.73 


64.25 


577.62 


36.74 


540.88 



^This figure was used as it is a little larger than the average nitrogen con- 
tent for the seeds, 34.70 milligrams, which would make even a greater fixation ap- 
pear at the harvest. 



The results of the separations are shown in Table 25. The figures 
were obtained in the same manner as those in Series 700. 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



537 



Table 25. — Nitrogen Separations: Series 600 (Cowpeas) 
(Milligrams per jar of five plants) 



Harvest 


Lab. 

Nos. 


Part 


Insoluble 
nitrogen 


Total 

soluble 
nitrogen 


P.T.A. 

nitrogen 


Other 

nitrogen 


Total 
nitrogen 


1 


611-613 
614-616 
617-619 


Tops 
Roots 
Nodules 


15.83 
6.92 


11.62 
2.13 


3.68 
.74 


7.94 
1.39 


27.45 
9.05 


2 


621-623 
624-626 
627-629 


Tops 
Roots 
Nodules 


23.73 
9.18 
7.54 


21.76 
1.89 
2.26 


5.88 
.42 
.92 


15.88 
1.47 
1.34 


45.49 

1L07 

9.80 


3 


631-633 
634-636 
637-639 


Tops 
Roots 
Nodules 


58.67 
11.18 
13.69 


32.54 
5.32 

4.83 


10.55 
1.90 
1.22 


21.99 
3.42 
3.61 


91.21 
16.50 
18.52 


4 


641-643 
644-646 
647-649 


Tops 
Roots 
Nodules 


97.82 
2L54 
26.74 


93.47 

7.61 

15.59 


32.49 
2.51 
9.08 


60.98 
5.10 
6.51 


191.29 
29.15 
42.33 


5 


651-653 
654-656 
657-659 


Tops 
Roots 
Nodules 


216.90 
52.35 
32.36 


226.50 
19.87 
31.89 


58.50 

5.17 

20.10 


168.00 
14.70 
1L79 


443.40 
72.22 
64.25 



The results of this series are also presented in a graph (Plate 
XVII). The curve of the soluble nitrogen does not show a decrease, 
possibly because the change upward had taken place before the end of 
the first fourteen days, when the first data were taken, as cowpeas 
show an early fixation of nitrogen and develop extremely rapidly un- 
der normal conditions. The same general tendencies hold thruout this 
series as in the others in respect to the increase of the soluble and 
insoluble nitrogen. When the soluble and the insoluble nitrogen 
ratios are considered, the results in general agree very closely in all 
the series regardless of time of growth and kind of legume. 



Discussion of Tables 

The results of the total nitrogen determinations of the four series 
show that, as an average of eighteen harvests, 74 percent of the nitro- 
gen of the cowpeas and soybeans Avas in the tops, the remaining 26 
percent being divided between the roots and the nodules. The figures 
show that in the first periods most of the 26 percent was in the roots, 
while later the nodules in some cases contained 18 of the 26 percent. 
In nine out of seventeen harvests, the nodules contained more nitro- 
gen than the roots of the same plants. 

The data showing the average daily fixation of nitrogen for five 
plants in the various series during the different growing periods are 
presented in Table 26. 



538 Bulletin No. 179 [March, 

Soluble ^nd Ih/bOL uBle Nij fioge n 

IN Tof>5,f^oor::> fjND NodulE^ of (_ o v^ -H E 'j i> 
J] IF FE TfE^y T f^EFfiOnS OrUEVELOfr/ENT 

Sfir /fcS GOO 

Legend 

Soluble, n In50luBl^^ 



cBM 



Tc 



0?=5 



ffoc 




J 




J J 



,4D^^ 2SJ]^ 3011c. ^iH--- 5,5 i7. 

Nodules 

Plate XVII 



1915] 



A Biochemical Study of Nitrogen in Certain Legumes 



539 



Table 26. — Average Daily Fixation of Nitrogen in All Series 



Series 


Periods 


Milligrams per 


in days 


jar of 5 plants 




0-38 


1.87 


100 

(Soybeans) 


38-53 


9.72 


53-60 
60-67 


19.84 
6.16 




67-74 


(-17.69) 




0-14 


.00 


500 


14-22 


1.51 


(Soybeans) 


22-30 


6.88 




30-41 


11.50 




0-12 


.00 


700 


12-23 


.62 


(Soybeans) 


23-31 


3.06 




31-42 


5.43 




0-14 


.24 


600 
(Cowpeas) 


14-22 


3.10 


22-30 
30-41 


7.73 
14.02 




41-58 


17.44 



The results included in Table 26 seem to indicate that the period 
during which the greatest total accumulation of atmospheric nitrogen 
takes place occurs between the fortieth and sixtieth days and coincides 
closely with the period just previous to seed formation. The greatest 
rate of increase of fixation and assimilation in these .series occurred in 
the early periods of growth. A comparison of Series 100 with the 
other series indicates that the growth of the plant is closely related to 
the rate and the amount of nitrogen fixed. The plants of Series 100 
grew much slower than the others. Those of Series 500 and 600 made 
the greatest growth in a given period, having had the advantage of the 
most favorable growing season, more especially for the cowpea, which 
requires a higher temperature than the soybean for optimum growth. 

The fixation in Series 600 for the whole period of fifty-eight days 
was 540.88 milligrams per five plants, or an average daily fixation of 
9.32 milligrams. The greatest fixation during any one period, as is 
evident from Table 26, occurred with the soybeans in Series 100 be- 
tween the fifty-third and sixtieth days, Avhen the daily average was 
19.84 milligrams per five plants, or nearly 4 milligrams per plant per 
day. If this figul*e is calculated to an acre basis, allowing a stand of 
four beans per square foot, an accumulation equivalent to one and 
a half pounds of nitrogen per acre per day is shown. 

The average percentages of soluble nitrogen in the four series 
in terms of total nitrogen in the particular part of the plant, may 
be of some interest, altho it will be seen from the accompanying graphs 



540 



Bulletin No. 179 



[March, 



that the amount depends upon the stage of growth when the harvest 
is made. These percentages were as follows: 

Table 27. — Percentages of Soluble Nitrogen in Each Series 
AS an Average of All Harvests 

(On the basis of total nitrogen in the given part) 



Series 


Tops 


Boots 


Nodnles 


100 (Soybeans) 
700 (Soybeans) 
500 (Soybeans) 
600 (Cowpeas) 


35.9 
57.7 
41.2 
45.0 


34.7 
39.5 
32.4 
27.5 


42.3^ 

18.9 

8.5 

34.0 



^The nodules in this series were not filtered thru a diatomaeeous earth filter 
but thru an ordinary filter and are therefore not included in the average given 
in the conclusions. 

The figures for Series 100 represent soluble nitrogen obtained in 
a hot-water extract. Series 500 and 600 are comparable with the 
exception of one being soybeans and the other cowpeas. The great 
difference in the solubility of the nitrogen in the nodules is particu- 
larly noticeable. 

There was a gradual increase in the soluble nitrogen in the nodules 
of Series 600 from the first harvest to the last, the percentages on the 
basis of total nitrogen being 23, 26, 37, and 49. A fact not brought out 
in the figures showing the soluble nitrogen is that in Series 700 and 
500 an extremely high soluble-nitrogen content was found in the tops 
and the roots at the first harvest. In Series 700 the percentage in 
the tops was 74, in Series 500, 57 ; while in the roots in Series 700 
the percentage was 56, and in Series 500, 50. 

On the basis of total nitrogen, the percentage of Other nitrogen 
in each series, as an average of all harvests, was as shown in Table 28. 
Other nitrogen is the difference between the total soluble nitrogen and 
that precipitated by P.T.A. and NaOH. It has been shown that a 
part of this nitrogen consists of amino acids, but as yet the total 
amount is unknown. 

Table 28. — Percentages of Other Nitrogen in Each Series 
AS AN Average of All Harvests 

(On the basis of total nitrogen in the given part) 



Series 


Tops 


Eoots 


Nodules 


100 (Soybeans) 
700 (Soybeans) 
500 (Soybeans) 
600 (Cowpeas) 


25.1 
36.1 
30.7 
28.3 


29.3 

32.8 
23.1 
17.2 


18.2 

6.9 

16.6 



The percentages of nitrogen precipitated by P.T.A. were as shown 
in Table 29. The variations as regards the tops are not easily ex- 
plainable. Undoubtedly there is a larger error in the P.T.A. deter- 
minations than in the others. The nodules of Series 600 contained 



1915] A Biochemical Study of Nitrogen in Certain Legumes 541 

large amounts of nitrogen which were precipitated by this reagent, 
the percentages at the harvests, from the second to the last, on the 
basis of total nitrogen being 9, 7, 21, and 31. 

Table 29. — Percentages of P.T.A. Nitrogen in Each Series 
AS AN Average of All Harvests 

(On the basis of total nitrogen in the given part) 



Series 


Tops 


Boots 


Nodules 


100 (Soybeans) 
700 (Soybeans) 
500 (Soybeans) 
600 (Cowpeas) 


7.00 
21.10 

6.75 
13.40 


3.0 
6.6 
6.0 
7.3 


l.i) 

1.4 

17.0 



The nitrogen obtained hy distillation with sodinm hydroxid ap- 
parently is not precipitated by P.T.A., as the percentage of Other 
nitrogen decreases without exception when sodium hydroxid is used. 
However, no definite conclusions can be drawn regarding the use of 
sodium hydroxid. 

CONCLUSIONS 

PART I 

1. The experiments reported show conclusively that the cowpea 
and the soybean utilize atmospheric nitrogen thru their roots and 
not thru their leaves. No combined nitrogen could have been assimi- 
lated in these gas experiments. 

PART II 

2. The total nitrogen determinations show that about 74 per- 
cent of the nitrogen of cowpeas and soybeans at the time of harvest is 
in the tops, while the remainder is distributed between the roots and 
the nodules. In the earlier periods the roots contain the larger part, 
w^hile later they contain much the smaller part. 

3. The percentage of soluble nitrogen in soybeans and cowpeas 
varies with the different parts of the plant and with the period of 
growth. In these experiments the soluble nitrogen, as an average, 
constituted in the tops about 45 percent of the total nitrogen; in 
the roots, 34 percent ; in the nodules of the soybeans, 14 i)ercent, and 
in the nodules oi the cowpeas, 34 percent. 

4. Phosphotungstic acid usually precipitates some form of nitro- 
gen. In some cases the amounts precipitated vary widely, while in 
others the agreement is close. In these series the nitrogen precii)itated 
by phosphotungstic acid averaged in the tops of both soybeans and 
cowpeas about 12 percent of the total nitrogen; in the roots. 5.5 per- 
cent ; in the nodules of the soybeans 1 percent, and in the nodules of 
the cowpeas, 17 percent. 



542 Bulletin No. 179 [March, 

5. Other forms of soluble nitrogen than those precipitated by 
phosphotungstic acid and sodium hydroxid occur. In these series 
they constituted as an average in the tops of both soybeans and cow- 
peas about 68 percent of the soluble nitrogen ; in the roots, 77 per- 
cent; in the nodules of soybeans, 89 percent, and in the nodules of 
cowpeas, 53 percent. 

6. Fixation takes place at a very early period in the growth of 
the seedling — sometimes within fourteen days. It is rapid in some 
cases, especially with cowpeas. 

7. Plants grown under the conditions of these experiments con- 
tain no ammonia, nitrites, or nitrates, as measured by the most ac- 
curate chemical methods. 



It is fully recognized that this work is incomplete, yet it is hoped 
that the study may aid in stimulating interest in some of these funda- 
mental problems. The lack of development of the various chemical 
methods used is partially responsible for some of the difficulties ex- 
perienced in their application. The survey presented of the chemical 
literature indicates a scarcity of existing knowledge regarding the 
more fundamental problems concerning nitrogen fixation. 

The biological resume points clearly to the need of more extended 
research along these lines. This is strikingly noticeable in the bac- 
teriological studies which have been undertaken with B. radicicola. 
Few cases are on record in which the authors actually proved out 
their cultures at the termination of their investigations by inocula- 
tion of a legume of the kind from which the organism originally came. 



The author takes this opportunity to express his gratitude to 
Professors C. G. Hopkins and J. H. Pettit for the many valuable sug- 
gestions they have so kindly given him. 



BIOGRAPHICAL SKETCH 

Albert Lemuel Whiting was born in Stoughton, Massachusetts, 
May 12, 1885. He secured his common-school education in the public 
schools of that town and then spent one jeav in mechanical work be- 
fore entering the Massachusetts Agricultural College in the fall of 
1904. From this institution he received the degree of bachelor of 
science in June, 1908. He immediately accepted a position as assist- 
ant agronomist at the Rhode Island Agricultural Experiment Sta- 
tion, where he also instructed in veterinary science in the College. 
At the same time he served as secretary of the Rhode Island Experi- 
mental Union. While there he also took graduate student work in 
agronomy and botany and in June, 1910, received the degree of 
master of science. In the fall of 1910 he was granted a fellowship 
in agronomy in the University of Illinois, which he held during 1910- 
12. He is now associate in soil biology in that institution. 

Dr. Whiting is a member of the following societies : Q.T.V. ; Alpha 
Chi Sigma ; the Illinois chapter of Sigma Xi ; Society of American 
Bacteriologists ; the American Society of Agronomy ; the American 
Chemical Society; and the American Society for the Promotion of 
Science. 



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